HSS-2305 Final Exam Notes

8. Gene Transcription and Translation

Sense VS Antisense:
1.​ Sense Strand:
-​ Also called the coding strand.
-​ Its sequence is identical to the newly synthesized RNA, except:
-​ Thymine (T) in DNA is replaced by Uracil (U) in RNA.
2.​ Antisense Strand:
-​ Serves as the template strand for RNA synthesis.
-​ RNA is synthesized as complementary to this strand.
Transcription Overview:
Process of synthesizing RNA from a DNA template. Enzyme involved = RNA polymerase
Steps:
1.​ Binding: RNA polymerase binds to the DNA at a specific site (promoter region)
2.​ Initiation: Transcription starts at the promoter.
3.​ longation: RNA polymerase incorporates nucleotides complementary to the DNA template strand.
4.​ Direction: RNA is synthesized in the 5’-to-3’ direction, using the 3’-to-5’ template strand.

Prokaryotic vs Eukaryotic Transcription

Prokaryotes:
One type of RNA polymerase transcribes all RNA types (mRNA, rRNA, tRNA).

Eukaryotes:
Three RNA polymerases:
-​ RNA Polymerase I: Transcribes rRNA.
-​ RNA Polymerase II: Transcribes mRNA and some snRNA.
-​ RNA Polymerase III: Transcribes tRNA and 5S rRNA.
★​ Reads the 3’-to-5’ template strand and synthesizes RNA in the 5’-to-3’ direction.

EUKARYOTES:

Transcription Factors (TFs)

Combine with RNA Polymerase II at the promoter region to form a preinitiation complex. Regulate gene
expression by initiating transcription.

Types:
1.​ General Transcription Factors (GTFs):
-​ Required for transcription by RNA Polymerase II.
-​ Includes: TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH.
-​ Assemble at the core promoter.
2.​ Specific Transcription Factors:
 -​ Regulate gene expression for specific genes.
TFIID: Unique GTF involved in transcription by RNA Polymerase II.
Components:
●​ TATA-Binding Protein (TBP):
○​ Recognizes and binds to the TATA box in the promoter region.
●​ TBP-Associated Factors (TAFs):
○​ Assist TBP in initiating transcription.
Function: Initiates the assembly of the transcription machinery (other GTFs and RNA Polymerase II).

Gene Promoter
-​ Definition: A DNA region where RNA polymerase binds to initiate transcription.
-​ Key Features:
-​ Determines which DNA strand serves as the template (antisense strand becomes the
mRNA sequence).
-​ Length: 100–1000 base pairs.
-​ Contains core and proximal promoter elements.
Core Promoter
●​ Function: Responsible for basic transcriptional activity.
Key Elements:
1.​ TATA Box:
-​ Consensus Sequence: TATAWAWR (Y=C/T, W=A/T, R=A/G).
-​ Present in ~20% of eukaryotic promoters.
-​ Recognized by TATA-Binding Protein (TBP), part of TFIID.
-​ Forms the preinitiation complex.
2.​ B Recognition Element (BRE):
-​ Flanks the TATA box in some promoters.
-​ Recognized by transcription factor TFIIB.
3.​ Initiator Element (Inr):
-​ Overlaps the TSS.
-​ Consensus Sequence: YYANWYY (A = start site, +1).
-​ Recognized by TBP-Associated Factors (TAFs).
4.​ Downstream Promoter Element (DPE):
-​ Located ~28–32 nucleotides downstream of the TSS.
-​ Consensus Sequence: RGWYV(T).
-​ Also recognized by TAFs.

Proximal Promoter
●​ Function: Enhances transcriptional activity of the core promoter and regulates transcription
frequency.
Key Elements:
1.​ CAAT Box:
-​ Recruits transcription factors to improve RNA polymerase II binding efficiency.
2.​ GC Box
-​ GC-rich regions bound by transcription factors like SP1
 -​ Activates genes, including constitutively expressed housekeeping genes.
3.​ Specific Response Elements:
-​ Respond to environmental or cellular signals.
-​ Examples:
-​ HRE (Hormone Response Element): Binds hormone receptors like glucocorticoid
receptors.
-​ CRE (cAMP Response Element): Binds CREB, responding to cAMP levels.

Enhancer Regions
●​ Definition: DNA sequences (50–1500 base pairs) that enhance transcription.
●​ Function:
○​ Bind transcription factors (TFs) and coactivators.
○​ Interact with the promoter region to enhance transcription by stabilizing the preinitiation
complex.

STEPS of Eukaryotic Transcprition:

1.​ Pre-Initiation Complex (PIC)

A large complex (~100 proteins) required for the transcription of protein-coding genes in eukaryotes.
●​ Functions:
a)​ Positions RNA Polymerase II at transcription start sites (TSS).
b)​ Denatures DNA to form a transcription bubble.
c)​ Aligns the DNA template in the RNA Polymerase II active site for transcription.
d)​ Facilitates binding of transcriptional activators (specific TFs) via coactivators to the PIC.
Formation of PIC:
1.​ TFIID Binding:
●​ Components:
○​ TBP (TATA-Binding Protein): Recognizes and binds the TATA box in the core (or
proximal) promoter.
○​ TAFs (TBP-Associated Factors): Assist in TFIID function.
●​ Role: First step in PIC assembly.

2.​ TFIIA and TFIIB Binding:

●​ TFIIA:
○​ Stabilizes the PIC.
●​ TFIIB:
○​ Provides a specific binding site for RNA Polymerase II.
3.​ RNA Pol II and TFIIF Binding:
●​ RNA Polymerase II, in complex with TFIIF, binds to TFIIB.
●​ TFIIF: Aids in transcription elongation.
4.​ TFIIE and TFIIH Binding:
●​ TFIIE: Facilitates the binding of TFIIH.
●​ TFIIH:
○​ Contains three enzymatic subunits:
 ■​ Kinase Activity: Phosphorylates RNA Pol II’s tail (CTD) to trigger
elongation.
■​ Two DNA Helicases: Unwind DNA at the promoter, forming a 13-bp
transcription bubble (requires ATP).

2.​ Initiation of Transcription

Formation of the first phosphodiester bond between the first two RNA nucleotides.
Steps
1.​ RNA Polymerase II slides along the DNA template, elongating the RNA strand.
2.​ Transcription factors of the PIC dissociate, except TFIID (remains bound for additional PIC
assembly).
3.​ Elongation
★​ Process: Extension of the mRNA transcript using the DNA template strand.
★​ Rate: RNA Polymerase II incorporates 20–50 nucleotides per second.
Key Features:
a)​ Transcription Bubble:
-​ Unwound DNA region of ~13 base pairs (bp).
-​ Front of RNA Pol II: DNA unwinds, creating positive supercoils.
-​ Behind RNA Pol II: DNA rewinds with negative supercoils.
-​ Topoisomerases relieve supercoiling stress.
b)​ DNA-RNA Hybrid:
-​ Stabilized by a hybrid region of ~8–9 bp between the DNA template and the nascent
RNA strand.
-​ RNA synthesis occurs in the 5′ to 3′ direction (or 3′ to 5′ on the DNA template).
c)​ Nucleotide Incorporation:
Example:
-​ ATP pairs with the T (thymine) on the DNA template (via hydrogen bonds).
-​ 3′ OH of the previous nucleotide binds to the 5′ α-phosphate of the incoming nucleoside
triphosphate.
-​ Pyrophosphate (PPi) is released and hydrolyzed to 2 inorganic phosphates (Pi), making the
reaction irreversible.

Elongation Factors:

P-TEFb:

●​ Phosphorylates RNA Polymerase II’s tail (CTD) at Ser-2.

●​ Recruits proteins for elongation and RNA modifications (e.g., splicing, polyadenylation).

ELL & TFIIF:

●​ Minimize nonspecific interactions between RNA Polymerase II and DNA.

●​ Prevent RNA Polymerase II from pausing during elongation.

TFIIS:
 -​ Helps RNA Polymerase II resume transcription after pauses.
-​ Proofreads and corrects RNA errors during elongation.

4.​ Termination
In Prokaryotes:
-​ Well-defined termination sequences guide transcription termination.
In Eukaryotes:
-​ No well-defined termination sequences.
-​ Termination is linked to the Polyadenylation Signal Sequence (PAS):
-​ Typically AAUAAA in the RNA transcript.
-​ Located well past the coding region.
Steps in Termination:
1.​ Cleavage: The new transcript is cleaved downstream of the PAS.
2.​ Polyadenylation: Template-independent addition of adenosines (Poly-A tail) to the 3′ end of the
RNA. Final length of the mRNA is determined during this processing.

9. Gene transcription and translation

Transcription: synthesis of complementary RNA from DNA template in the nucleus (DNA→mRNA)
●​ Start: DNA template strand (provides sequence for RNA synthesis)
●​ Required components:
○​ RNA polymerase II → enzyme that transcribes DNA to mRNA and some snRNA
○​ Transcription Factors → proteins that control gene to turn on/off. TFs can activate or
inhibit transcription
●​ End product: mRNA
Primary Transcript (pre-mRNA or hnRNA): initial RNA molecule synthesized during transcription in
eukaryotic cells
-​ Unprocessed: contains exons and introns
-​ Same length as DNA template strand
C-terminal domain: is essential for RNA processing, serving as a platform to recruit and organize
RNA-processing machinery during transcription

Processing of pre-mRNA
1) Capping (5’Caps)
●​ Adds protective cap to mRNA during transcription
○​ 5’ CAP (7’Methylguanosine cap)
■​ Protects mRNA from degradation by exonucleases
●​ Exonucleases: enzyme that breaks down mRNA
■​ Aids in transport of mRNA out of nucleus
■​ Plays important role in initiation of translation (ribsomes recognize)
●​ STEPS
1)​ Remove terminal phosphate from 5’ end
2)​ GMP added in inverted orientation which links 5’end of mRNA and GMP
 a)​ This creates 7-methylguanine (5’ CAP)
3)​ Methyl groups added.
a)​ Stabilizes and marks ready for translation
2) Polyadenylation (3’ poly (A) tail)
●​ Poly(A) tail enzymes recruited by phosphorylated CTD:
○​ Protects mRNA from premature degradation by exonucleases
●​ STEPS
1)​ Endonuclease cleaves primary RNA which creates a new 3’ end where poly (A)
tail will be added
2)​ Adenosine residues added to new 3` end by Poly A polymerase
3)​ 200-250 adenosine residues added to poly (A) tail. Poly(A)-binding proteins
attach and stabilize the mRNA and help with nuclear export to cytoplasm
3) RNA Splicing (exons+introns)
●​ Exons: parts of the gene that contribute to mature RNA product and later translated
(remain after splicing)
●​ Introns: Not included in mature RNA product → are spliced out
●​ Splice Sites: Break starts at 5’ and ends at 3’end
●​ Exon-intron boundaries have highly conserved sequences that are crucial for accurate
splicing
○​ 5' splice site: G/GU marks the start of the intron.
○​ 3' splice site: AG/G marks the end of the intron.
○​ Exonic splicing enhancers (ESE): help recruit Small Nuclear Ribonuclear
proteins and found within exons
■​ snRNPs: core part of the spliceosome
●​ Consist of: small nuclear RNAs •
○​ Ex. U1, U2, U4, U5, U6
●​ 12+ associated proteins
○​ Ex. U1, U2, U4, U5, U6
●​ Function: Splicing, Ligating pre-RNA into final mRNA product
●​ Splicesome: composed of snRNPs and associate proteins. Revomes introns and connects
extrons

RNA SPLICING

1. Recognition and Binding

●​ U1 snRNP:
○​ Attaches to the 5' splice site of the pre-mRNA.
○​ The nucleotide sequence of U1 snRNA is complementary to the 5' splice site sequence
(G/GU).
●​ U2AF (U2 Auxiliary Factor):
○​ Binds to the polypyrimidine tract and the 3' splice site.
○​ Attracts U2 snRNP.

2. Branch Point Formation

 ●​ U2 snRNP:
○​ Attaches to the branch point sequence within the intron.
○​ Causes a specific adenosine residue to bulge out, forming the branch point of the
"lariat" intron loop.

3. Spliceosome Assembly

●​ U4/U6 and U5 snRNPs:

○​ Bind to the pre-mRNA.
○​ U4 and U6 snRNAs are initially paired together.
○​ U4 snRNA is stripped away, allowing U6 snRNA to pair with U2 snRNA.

4. Catalytic Steps of Splicing

●​ U6 snRNP:
○​ Displaces U1 snRNP and binds to the 5' splice site.
○​ Functions as a ribozyme, catalyzing the splicing reactions:
■​ First cleavage reaction (Rx1):
■​ Cleavage at the 5' splice site.
■​ Produces:
■​ A free 5' exon (Exon 1) held in place by U5 snRNP.
■​ A lariat intron–3' exon intermediate.
■​ Second cleavage reaction (Rx2):
■​ Cleavage at the 3' splice site.
■​ Produces:
■​ The excised lariat intron.
■​ The ligated exons (Exon 1 and Exon 2).

5. Completion

●​ Release of snRNPs:
○​ snRNPs dissociate from the pre-mRNA.
○​ They reassemble at other introns for further splicing.

This organized sequence highlights the roles of snRNPs and associated factors in pre-mRNA splicing.

★​ Alternative Splicing: allows single gene to produce multiple different mRNA transcripts= various
different proteins from same gene sequence → greater diversity of proteins

Different Types of Transcipts:

rRNA = ribosomal rRNA

-​ Catalyzes amino acid covalent linking during translation

-​ Provide structural support
-​ > 80% of cells RNA is rRNA
 -​ Large ribosome subunit (60S)→ 5S, 5.8S, 28S
-​ Small ribosome subunit (40S)→ 18S

Synthesis:

-​ Clusters of rRNA genes are arranged in tandem: this allows for efficient production of rRNA
-​ Nontranscribed spacers (NTS)= Separates transcription units in a ribosomal gene cluster and do
not transcribe/ code RNA (act as spacers)

rRNA transcribed by:

-​ RNA Pol I (28S, 18S, 5.8S – parts of both the 60S and 40S subunits)
-​ • RNA Pol III (5S – smallest part of the 60S subunit) – note that 5S is not made in the clusters and
is created outside of the nucleoli
-​

rRNA PROCESSING → takes place in nucleoli (nucleolus)

Key components of the nucleus

1.​ Fibrilllar cente: contains rDNA

2.​ Dense fibrillar component: contains the pre rRNA transcripts
3.​ Granular component: contains ribosomes at various levels of assembly

Methylation + Pseudouridylation = play crucial roles in the structural stability and function of ribosomes

Small nucleolar ribonucleoproteins (snoRNPs ): consist of small nucleolar RNAs (snoRNAs) and
associated proteins. Play active role in processing pre RNA in nucleolus to help the structural stability and
function of ribosome (Act as guides for site-specific chemical modifications of rRNAs.)

-​ Box H/ACA snoRNA determine which uridines will convert to pseudouridines.

-​ Box C/D snoRNA determine which nucleotides in the pre RNA will have ribose methylated→
crucial for ribose function

tRNA = transfer RNA

-​ Matches mRNA nucleotide code to amino acids for polypeptide during translation
-​ Anti codon binds to specific amion acid
-​ Where are they transcribed??? → tRNA genes, nontranscribed spacer, tRNA promoter
sequences, tRNA precursor is processed and trimmed

Translation: synthesis of proteins in the cytoplasm using information encoded by mRNA

●​ Start: mRNA
●​ Required components:
○​ Ribosomal RNA (rRNA) and ribosomal proteins
 ○​ Transfer RNA (tRNA)
●​ End product: polypeptide
Codon: nucleotide triplets, matched with amino acids.
-​ Ex: UGU → Cysteine. Use a table to using the letters of the triplet
-​ 20 amino acids but out of the 64 codons only 61 encode amino acids
-​ Degenerate code: multiple codons can code for the same amino acid during protein synthesis.
-​ Ex: UUA, UUG, CUU
Anti-codon: Sequense on tRNA that are complementary to the codon
-​ A → U
-​ G → C
-​ Ex: Methionine: The codon is AUG, and the anticodon is UAC

tRNA Isoacceptors: tRNAs with unique anticodons that can bind to the same amino acid.

★​ There are 61 codons but only 50 tRNA → possible because of wobble pairing
Wobble Pairing
➔​ Refers to flexibility in base pairing at the third position of a codon
◆​ EX: G in the anticodon can pair with either U or C in the codon
➔​ The wobble position generally pairs with either:
◆​ A purine (G or A).
◆​ A pyrimidine (U or C).
★​ Codon Degeneracy + Wobble Pairing reduce DNA mutations

tRNA Formation:
1.​ Aminoacyl-tRNA Synthetases (AARS): Enzymes that attach a specific amino acid (AA) to the 3'
end. Exactly 1 for each 20 AA
2.​ Process: ATP required
a.​ Step 1: Amino acid is activated
b.​ Step 2: Activated amino acid is transferred to tRNA → final product: aminoacyl-tRNA
Translation process (initiation, elongation, termination in eukaryotic)

STRUCTURE OF RIBOSOME for tRNA :

●​ A site (Aminoacyl): Entry point
●​ P site (Peptidyl): Holds the growing peptide chain.
●​ E site (Exit): Where uncharged tRNA exits the ribosome.

1.​ Initiation → where the ribosome assembles on the mRNA and prepare
Step 1: Binding of Initiation Factors
-​ About 12 eukaryotic initiation factors (eIFs) bind to the small ribosomal subunit (40S)
-​ eIFs: stabilize and prepare the ribosomal subunit for mRNA binding
Step 2: Loading of Initiator tRNA-Met
-​ initiator tRNA (tRNA-Met) enters the P site
-​ This occurs in association with eIF2-GTP, which provides energy
Step 3:
 -​ The 43S complex is formed when the ribosome binds to the 5' cap of the mRNA
-​ Several eIFs assist in this process:
-​ eIF4E binds the 5' cap of the mRNA.
-​ eIF4A unwinds double-stranded RNA regions in the mRNA, ensuring it’s ready for
translation.
-​ eIF4G connects the 5' cap to the 3' poly(A) tail, creating a circularized mRNA structure
that enhances stability and translation efficiency.
Step 4: Scanning for the Start Codon
-​ The 43S complex scans to locate the start codon (AUG)
-​ In eukaryotes, the Kozak sequence (5' ACCAUGG 3') helps guide the ribosome to the correct
AUG codon.
Step 5: Assembly of the Full Ribosome
-​ Once start is located eIF2-GTP is hydrolyzed (GTP → GDP), releasing energy to drive the
process.
-​ The large ribosomal subunit (60S) joins the complex, forming the 80S ribosome.
-​ All initiation factors are released, leaving a fully assembled ribosome

2.​ Elongation → where amino acids are sequentially added to a growing polypeptide chain
Step 1:
-​ The second aminoacyl-tRNA binds to the elongation factor eEF1α-GTP.
-​ Complex is brought to the A site
Step 2:
-​ Aminoacyl-tRNA is placed into the A site of ribosome
-​ GTP is hydrolyzed, converting it to GDP and releasing eEF1α-GDP
-​ Only the tRNA with the correct anticodon for the mRNA codon in the A site can trigger
conformational changes
Step 3:
-​ Amine nitrogen of the amino acid on the tRNA in the A site performs a nucleophilic attack on the
carbonyl carbon of the amino acid on the tRNA in the P site.
-​ The enzyme peptidyl transferase catalyzes the formation of a peptide bond between the two
amino acids
-​ No external energy is required
Step 4:
-​ The ribosome moves three nucleotides (one codon) along the mRNA in the 5'-3' direction, a
process called translocation.
-​ During this movement:
-​ The tRNA in the A site (now carrying the growing peptide chain) shifts to the P site.
-​ The tRNA in the P site (now uncharged) moves to the E site.
-​ This movement is driven by conformational changes in eEF2 upon GTP hydrolysis.
Step 5:
-​ Uncharged (deacylated) tRNA in the E site exits the ribosome, leaving the E site empty
Step 6:
-​ Repeat steps
-​ Each cycle adds one amino acid to the polypeptide chain.
 3.​ Termination → Termination occurs when the ribosome encounters one of the three stop codons
-​ UAA, UAG, UGA
-​ Requires release factors which recognize the stop codon
-​ eRF1: Recognizes all three stop codons and interacts directly
-​ eRF3: A GTPase that provides energy for the release process by hydrolyzing
GTP.

*Polyribosomes: multiple ribsomes on mRNA→ allows simultaneous translation

-​ Increase the rate of protein synthesis

10. Control of Gene Expression

Nucleus
-​ Site of transcription
-​ Nucleolus → substructure in the nucleay that synthesizes rRNA and ribosomes
-​ Chromatin → tightly packed DNA
1.​ Euchromatin: loosely packed + more accessible to transcription + gene rich
2.​ Heterochromatin: tightly packed + transcriptionally silent + gene poor
-​ Proteins used for transcription transported into nucleus via nuclear pore
-​ RNA products for translation transported out of nucleus via nuclear pore
Nuclear Envelope
-​ Double membrane
-​ Outer continues with RER
-​ Protein synthesis + transport
-​ Inner membrane
-​ Supported by nuclear lamina that provides support and chromatin anchoring
Nuclear Pore Complex (NPC)
-​ Composed of nucleoporins. Acts as a gateway for molecules moving from nucleus→ cytoplasm
for RNAs and proteins
-​ Nucleoporins have FG domains → form hydrophobic mesh that blocks free diffusion of large
molecules
Transport of Protein Into Nucleus
-​ Proteins contain specific AA that indicate if they can transport
1.​ Nuclear Localization Signal (NLS) → direct import into nucleus
2.​ Nuclear Export Signal (NES) → direct export out of nucleus
-​ Transport Proteins
1.​ Importins →binds to NLS and into nucleus
2.​ Exportins →binds to NES and out nucleus
Transport of mRNA Across Nuclear Envelope
-​ mRNA Export
-​ mRNAs transported as ribonucleoproteins (RNPs)
 -​ Associated proteins interact with FG domain of nucleoporins for selective transport
-​ Only mature mRNAs (fully processed) are exported
-​ Nxf1 and Nxt1 Dependent and not exportin or RanGTP dependent
mRNA are transported as RNPs via Nxf1 and Nxt1

Gene Regulation
1.​ Transcriptional level control
2.​ Processing level control
3.​ Translational level control
4.​ Post translational control

1. Transcriptional Control → Transpcrition Factors (TFs)

a)​ General transcription factors → bind to core promoter sites, associate with RNA Pol
-​ TBP (TATA binding protein), TAF, TFIIA-H (component of general transcription
complex)
b)​ Sequence-specific transcription factors → bind to regulatory sites of particular gene
-​ Activators = stimulate transcription
-​ Repressors = inhibit transcription
- Each TF regulates many different genes and each gene regulated by many different TF

Structure of TF:
-​ DNA-binding domain = binds to specific sequences of base pairs on DNA
-​ Bind to DNA with
-​ Van der Waals forces
-​ ionic bonds
-​ hydrogen bonds
TYPES OF DNA-BINDING DOMAINS
1.​ Zinc fingers
-​ They consist of a combination of (Cys) and (His) residues that coordinate the zinc ion
-​ Binds in major grooves in DNA and recognizes specific base sequences.
2.​ Helix-Loop-Helix (HLH)
-​ This motif consists of two α-helices connected by a flexible loop often proceeded by very
basic AAs (lys, arg, his)
-​ These amino acids play a crucial role in DNA binding due to their polar and positively
charged side chains
-​ Dimerization
3.​ Leucine Zipper
-​ Series of α-helix chains. Leucines form a "zipper" that facilitates dimerization between
two transcription factors
-​ binds to specific DNA sequences, typically at basic regions

-​ Activation domain = site of interaction with other proteins

-​ Dimerization domain = This domain promotes binding with another protein of identical or
similar structure
 ★​ Transcription factors bind to response elements
Response Elements are found in…
1.​ Proximal promoters: have common consensus sequences for general transcription factors, ex:
• CAAT box (I.e. 5’-GGCCAATCT-3’)
• GC box (I.e. 5'-GGGCGG-3’)
2.​ Distal promoter elements: located farther upstream from the TSS
-​ do not typically contain fixed consensus sequences like proximal promoters
-​ but may include enhancers or silencers that interact with transcription factors to modulate gene
expression based on cellular conditions

Enhancer site: are regulatory DNA sequences that bind transcription factors (activators) and co-activators
(e.g., mediators)
Co-activators:

●​ Definition: Proteins that increase gene expression.

●​ Key Feature: They do not directly bind to DNA, so they are not transcription factors.
●​ Function: Co-activators assist transcription by either:
1.​ Directly interacting with the transcription machinery.
-​ Mediator Complex: A specific type of co-activator.
-​ Role: Interacts with RNA Polymerase II (a core part of the PIC).
-​ Function: Links activators (transcription factors) to the transcription machinery.
-​ Significance: Essential for the expression of nearly all protein-coding genes.
2.​ Modifying chromatin to make DNA more accessible for transcription factors.
-​ Purpose: Remodel chromatin to make specific regions of DNA accessible for
transcription factor binding.
-​ Process: Known as chromatin relaxation, achieved through:
-​ ATP Hydrolysis: Energy-driven alterations to nucleosomes, such as:
-​ Sliding: Moving histones to a new position along the DNA.
-​ Conformational Change: Altering the nucleosome structure.
-​ Variant Histone Exchange: Replacing standard histones with variants that
enhance transcription.
-​ Histone Displacement: Removing histones from DNA entirely.
-​ Key Players:
-​ Histone Acetyltransferases (HATs):
-​ Add acetyl groups to lysine residues in histones.
-​ Acetyl groups reduce the positive charge of histones.
-​ Weakens histone-DNA binding, exposing DNA for transcription.
-​ Chromatin Remodeling Complexes:
-​ Mechanically adjust chromatin to enhance promoter accessibility.

2. Processing Level Control → only processed mRNA can exit the nucleus
Different splice sites can be repressed or activated, it depends on nearby binding proteins:
 a)​ SR proteins
-​ Activate splice sites
-​ Bind to exon/intron splicing enhancers (ESE/ISE)
b)​ hnRNP (heterogeneous RNA = proteins)
-​ Repress splice sites
-​ bind to exon/intron splicing silencers (ESS/IS)
3. Translational Level Control → 5’ and 3’ untranslated regions (UTRs) of mRNA play an
important role in regulation

Regulatory Mechanism:
1.​ Initiation and progression of translation (2 categories)
a.​ Global Regulation (broad translation suppression)
-​ Definition: Affects the translation of all mRNAs in the cell simultaneously.
-​ Mechanism: Involves the phosphorylation of translation initiation factors, which inhibits their
function
-​ Ex: phosphorylation of elF2→ elF2 cannot exchange GDP for GTP no binding of tRNA to
mRNA= no translation
-​ When It Happens: This occurs under stress conditions, such as nutrient deprivation
b.​ Specific (Fine-tuned control of individual mRNAs)
-​ Definition: Affects the translation of specific mRNAs, often in response to specific signals or
cellular conditions.
-​ Mechanism
-​ Many mRNAs have untranslated regions (UTRs) with specific structures like stem loops
that act as regulatory elements.
-​ Proteins bind to these regions to regulate translation by either promoting or inhibiting
ribosome binding.
-​ Ex: ferritin translation is regulated by iron levels via IRP binding to the IRE
2.​ Localization of mRNAs to certain sites within the cell
Definition: Specific mRNAs are transported and confined to specific regions of the cytoplasm to enable
location-specific protein production.
Significance: This mechanism allows for precise spatial and temporal control of protein synthesis,
particularly important in cells with distinct regions (e.g., polarized cells).
●​ Localization signals in the 3' UTR act as zip code
●​ RNA-binding proteins to guide mRNA.
●​ Transport via microtubules and anchoring via microfilaments.
●​ The translation is blocked during transport, ensuring proteins are synthesized only at the correct
location.

This system is crucial for cells with functional domains, like polarized cells, enabling precise protein
targeting and cellular efficiency.

3.​ mRNA stability within the cytoplasm

-​ mRNA Stability: Determines how long mRNA is translated in the cytoplasm.
 -​ Poly(A) Tail: Starts at ~200 adenosines and is gradually shortened by deadenylase.
-​ Shorter tails = Less stability = Faster degradation.
-​ 3' UTR Regulation:
-​ Sequences in the 3' UTR regulate mRNA degradation.
-​ AU-rich elements (AREs): Destabilize mRNA by promoting deadenylation.
-​ miRNAs: Bind to the 3' UTR and recruit deadenylation factors, reducing mRNA stability.
-​ Unless protected mRNA with short (<30)/absent poly(A) tails are rapidly degraded
★​ RNA Interference (RNAi): is a biological process where small RNA molecules inhibit gene
expression by targeting and destroying specific mRNAs. Regulates protein production
EX:
-​ Small Interfering RNAs (siRNAs) in plants: designed siRNA can specifically and
effectively silence a disease-related gene
-​ MicroRNAs (miRNAs) in mammals and play a major role in the development of many
common diseases.
4.​ Translational Level Control
Protein degradation is a critical cellular process used to regulate protein levels and remove damaged,
misfolded, or unneeded proteins, ensuring proper cellular function.
Key players:
-​ Proteasomes: protein degrading machines, found in nucleus and cytosol
Process of degradation:

1.​ Ubiquitination: Proteins are tagged with ubiquitin by E1, E2, and E3 ligases.
2.​ Binding: Polyubiquitinated proteins bind to the proteasome cap.
3.​ Preparation: Ubiquitin is removed, and the protein is unfolded.
4.​ Degradation: The proteasome’s β subunits degrade the protein into small peptides.
5.​ Release: Peptides are released into the cytosol for further processing or reuse.

11+12 Gene-Environment Interactions - Epigenetics

Chromosomes:
-​ Packages large amounts of DNA into a small space.
-​ It contains a single, continuous piece of DNA
-​ Chromosomes exist as homologous pairs
Diploid Cells
-​ Human diploid cells contain 23 pairs of chromosomes (46 total chromatids).
-​ 22 pairs of autosomal chromosomes.
-​ 1 pair of sex chromosomes.
Haploid Cells
-​ Contain 23 unpaired chromosomes (single chromatid from one parent).
-​ Result of meiosis.
-​ Example: Gametes (sperm and oocytes)
Haploid cells: 23 chromosomes total. Diploid cells: 46 chromosomes (23 pairs) in total.

Chromatin: DNA + associated proteins.

Histones:
-​ Proteins that package DNA.
-​ Octamer structure: Composed of 8 proteins.
-​ Positively charged, interacting with negatively charged DNA.
Histone Tails:
-​ Located at the N-terminus.
Modified by enzymes to affect: Histone charge, DNA compaction, and Gene transcription.
Nucleosome: Basic unit of chromatin.
-​ Composed of:
-​ Histone core: Contains 2 copies each of H2A, H2B, H3, H4.
-​ DNA: 146 base pairs wrapped 1.65 times around the histone core.
H1 - linker histone: Binds to linker DNA and connects nucleosomes
Histone code hypothesis
-​ Activity of a chromatin region depends on the degree of chemical modification of histone tails
Epigenetics: Study of heritable changes in gene expression or phenotype that do not involve
changes in the DNA sequence.
-​ Involves functionally relevant modifications to the genome that:
-​ Turn genes on (activate) or off (silence).
-​ Potentiate or inhibit gene expression.
-​ Provides a means for genetic material to respond to environmental changes
-​ Critical to morphogenesis
-​ Ex: Yamanaka factors: can reprogram the epigenome to convert somatic cells into
induced pluripotent stem cells
What causes these changes???
-​ Nature: Inheritance and normal development
-​ Nurture: Diet, exercise, hormones, toxins
-​ PROOF: Agouti mouse model (fat different colour mouse when the gene was activated)
and twin study (different environment expures→ led to health diseases)

Mechanisms of Epigenetics
1.​ DNA Methylation: addition of methyl group on cysteine
-​ Silences gene expression
-​ No methylation of CpG: Gene is "on."
-​ Examples: Tissue-specific genes, and housekeeping genes.
-​ Methylation of CpG: Gene is "off."
-​ Examples: Genes not specific to tissue, silent DNA.
-​ Mechanism
-​ DNA Methyltransferases (DNMTs) → responsible for adding the methyl
-​ Knockout of DNMTs: Lethal in animals during early development.
-​ Overexpression of DNMTs: Linked to cancer (e.g., leukemia).
-​ Obtained methyl groups through diet

2.​ Histone Modification

 a)​ Methylation of Histones: Addition of methyl groups to lysine or arginine residues on histone
tails.
-​ Gene silencing marker: Example: Methylation of lysine 9 on Histone 3.
-​ Gene activation marker: Example: Methylation of lysine 4 on Histone 3.
Enzymes involved:
-​ Histone methyltransferases (HMTs/KMTs): Add methyl groups.
-​ Demethylases (KDMs/JmjC-domain): Remove methyl groups.
b)​ Acetylation of Histones: Addition of acetyl groups to lysine residues on histone tails.
-​ Removes positive charge of histone tails.
-​ Loosens histone-DNA complex, facilitating gene activation.
Enzymes involved:
-​ Histone acetyltransferases (HATs/KATs): Add acetyl groups. (loosens)
-​ Histone deacetyltransferases (HDACs/KDACs): Remove acetyl groups. (tightens)

3.​ Long Non-Coding RNA (lncRNA)

-​ lncRNA: Sequence-specific RNA molecules that guide protein complexes to specific chromatin
sites.
-​ It does transcriptional repression by targeting and modifying chromatin structure

Epigenetic Reprogramming = Erasure and remodeling of epigenetic marks during development.

Key Stages:
-​ Gametogenesis (formation of gametes - sperm and oocytes via meiosis):
-​ Imprint erasure.
-​ Expression occurs from 1 allele only in a parent-specific manner.
-​ Example: Maternal imprint turns the mother's gene "off", the father’s gene "on".
-​ Controlled by DNA methylation and histone modification.
-​ Occurs in less than 1% of genes (~80 genes)
-​ PROCESS:
-​ Process:
-​ Imprints are erased.
-​ New imprint is established based on the individual's sex (e.g., sperm =
paternal imprint).
-​ Imprint is maintained during fertilization and pre-implantation.
-​ Imprint persists in somatic cells for a lifetime.
★​ Imprinted genes are highly susceptible to diet, hormones, toxins due to single active copy.

-​ X-chromosome reactivation (females only).

-​ Parental imprinting (sex-specific).
-​ Pre-implantation: Post-fertilization stage as embryo develops into a blastocyst
-​ Genome-wide demethylation (except for imprinted/non-imprinted genes).
-​ Tissue-specific methylation patterns.
-​ X-chromosome inactivation (females only).
-​ Ensures only 1 X chromosome is active (like males).
 -​ Random silencing of one X chromosome in each cell, mediated by epigenetic
modification.
-​ Mechanism:
-​ XIST: Long non-coding RNA coats the X chromosome to be silenced.
-​ TSIX: Blocking protein protects the active X chromosome from
silencing.
-​ ~25% of X-chromosome genes escape inactivation (correspond to genes present
on the Y chromosome).
-​ Maintained across the lifespan.
★​ Diet, hormones, and toxins can affect: Gene expression, Imprinting processes, Methylation and
histone modification patterns in the epigenome.

EPIGENETICS AND DISEASE

-​ Adult Onset: Most cancers manifest in adulthood.
-​ Many cancers involve both DNA mutations and epigenetic changes.
Mechanisms of Epigenetic Modification in Cancer
1.​ DNA Methylation:
-​ Hyper-methylation (excess): Silencing tumor suppressor genes.
-​ Hypo-methylation (reduces): Activation of oncogenes.
-​ BALANCE IS KEY!
2.​ Histone Modification:
-​ Alterations in methylation and acetylation of histones can disrupt normal gene
expression.
-​ It can silence tumor suppressor gene
3.​ Disruption of Epigenetic Machinery:
-​ Abnormal activity of enzymes:
-​ DNA methyltransferases (DNMTs). → hypermeth → overactivity
-​ Histone deacetylases (HDACs). → silences tumor suppressor
4.​ Loss of Imprinting:
-​ Example: IGF-2 (Insulin-like Growth Factor 2).
-​ In Wilms Tumor, loss of imprinting leads to overexpression of IGF-2, causing
excessive signals for cell growth and contributing to tumour formation
-​ Both parental copies of genes = active → overexpression of growth factors

13. DNA Replication, Damage, Repair

DNA REPLICATION
-​ Copying of DNA
-​ Replication machinery is also used for DNA repair
-​ Human diploid cells have 46 chromosomes (gametes/haploid have 23)
Semi-Conservative Replication: Watson & Crick: Proposed model.
-​ Gradual strand separation of the double helix (hydrogen bonds broken, "zipper-like").
-​ Two daughter strands synthesized complementary to the parental templates.
-​ Each daughter duplex contains one parental strand.

Eukaryotic DNA Replication

S-Phase:
-​ DNA replication occurs in the “Synthesis” phase of the cell cycle.
Replicons:
-​ Many small portions of the eukaryotic genome are replicated (replicons) all at once
Origins:
-​ Each replicon has an initiation site.
Human cells: ~10,000–100,000 origins
-​ 10-15% replicons active during S-phase.

Replication Process
●​ Bi-Directional: Replication proceeds in both directions from the origin.
Replication Fork:
-​ Site of strand separation in the parental double helix.
-​ Nucleotides added to form new complementary strands.
-​ Two replication forks move in opposite directions.
Nuclear Structure:
-​ Replication Foci:
-​ Localized sites of active replicons (~50-250 foci/nucleus)
-​ Each foci contains ~10-100 active replication forks.

Prokaryotic DNA Replication

DNA Polymerase Holoenzyme:

●​ Synthesizes daughter strands (leading & lagging) simultaneously.
●​ Key enzymes:
○​ DNA Polymerase III (main replication)
○​ DNA Polymerase I (replaces RNA primer with DNA).

DNA Polymerase Requirements:

1.​ Template DNA strand.
2.​ RNA Primer to add nucleotides.
3.​ Synthesizes DNA in 5' → 3' direction.

Mechanism:
-​ the 3'OH group performs a nucleophilic attack on the 5'α-phosphate of incoming nucleoside
triphosphate.
Semi-Discontinuous Replication
-​ Leading Strand: Synthesized continuously toward the replication fork.
-​ Lagging Strand: Synthesized discontinuously away from the fork.
-​ Forms Okazaki Fragments, each requiring a small RNA primer.
-​ Function: Reduces mismatch errors.

Prokaryotic DNA Replication

Initiation

●​ Dna A: Recognizes origin of replication (OriC).

●​ Dna B Helicase:
●​ Unwinds DNA using ATP hydrolysis.
●​ Breaks hydrogen bonds between strands.
●​ Single-stranded DNA-Binding (SSB) Proteins: Stabilize unwound DNA.
●​ Primase: Synthesizes RNA primers for Okazaki fragments.
●​ DNA Polymerase III: Extends RNA primers as part of holoenzyme.

Elongation

●​ DNA Polymerase III Holoenzyme:

1.​ 2 core polymerases for leading and lagging strands.
2.​ β Clamps: Hold polymerase on DNA (1 for leading strand, 1 per Okazaki fragment).
3.​ γ Clamp Loader: Loads sliding clamp and interacts with helicase.
●​ Lagging Strand:
1.​ RNA primers extended by DNA Polymerase III.
2.​ Polymerase releases lagging strand upon encountering an Okazaki fragment.
3.​ DNA Polymerase I removes RNA primers (exonuclease) and fills gaps with DNA.
4.​ DNA Ligase seals nicks between Okazaki fragments.
●​ Replisome: Entire protein complex at the replication fork (DNA pol III holoenzyme, helicase,
primase, SSBs).

Supercoiling

●​ DNA Gyrase: Relieves supercoiling tension during replication.

Eukaryotic DNA Replication

Initiation

●​ Origin Recognition Complex (ORC):

 ●​ Recognizes origins of replication within replicons.
●​ Comprised of 6 proteins.
●​ Licensing Factors:
●​ Cdc6 and Cdt1: Recruit helicase.
●​ Helicase: Unwinds DNA, made of MCM proteins (MCM2-7).
●​ Pre-Replication Complex (Pre-RC):
●​ Assembles during G1 phase.
●​ Activated by protein kinases (Cdk, DDK) in S-phase.
●​ Prevents new Pre-RCs from forming during S-phase, ensuring replication occurs only
once per cell cycle.

Elongation

●​ Replication Fork:
●​ ORC, licensing factors, and helicase active during G1.
●​ Primase synthesizes RNA primers for Okazaki fragments.
●​ DNA polymerases extend RNA primers.
●​ Synchronization:
●​ Pre-RCs form only in M and G1 phases for the next cell cycle.

Supercoiling

●​ Type I Topoisomerases: Relax supercoiling by nicking one DNA strand (no ATP required).
●​ Type II Topoisomerases: Break and rejoin double-stranded DNA to introduce or remove
supercoils (ATP required).

Chromatin Structure

Nucleosome Dynamics:

-​ Replication machinery displaces nucleosomes as it moves along the DNA.

-​ Nucleosomes reassemble quickly on daughter strands.

Histone Recycling:

-​
Histones from parental chromosomes and newly synthesized histones contribute to
reassembly.
-​ (H3H4)₂ Tetramers: Remain intact.
-​ H2A/H2B Dimers: Separate and bind randomly to new or old tetramers.
★​ ~80 diseases caused by mutations in replication/repair proteins.
★​ EX→ Diseases associated with mutations in helicase proteins, leading to defects in unwinding
and DNA synthesis.
DNA REPAIR

High Fidelity DNA Replication

Spontaneous Mutation Rate:
-​ Low error rate for incorporating incorrect nucleotides during replication.
Nucleotide Selection:
-​ Incorporation depends on geometry: only the correct nucleotide forms a proper geometric
fit with the template strand.
-​ DNA polymerase binds the correct nucleotide in its binding pocket

Prokaryotic Mismatch Repair

Proofreading Function:
-​ DNA Polymerase I and III:
-​ Have 3’→5’ exonuclease activity.
-​ Reverse excision of mispaired nucleotides at the 3’ end during replication.
A similar function in eukaryotes is performed by DNA polymerase ε/δ.
Mechanism: Incorrect base pairs are removed and replaced to ensure high accuracy during DNA
synthesis.
DNA Damage
Susceptibility:
-​ DNA is highly susceptible to environmental damage from:
-​ Ionizing radiation.
-​ Chemicals.
-​ UV radiation.
-​ Thermal energy from normal metabolism.
-​ Results in ~10,000 base lesions per cell per day (<0.1% escape repair).

Repair Mechanisms:
1.​ Accurate nucleotide selection during replication.
2.​ Immediate proofreading by DNA polymerase.
3.​ Post-replicative mismatch repair to correct errors missed during replication.

Eukaryotic Nucleotide Excision Repair (NER)

Purpose:
-​ Removes bulky DNA lesions caused by:
-​ UV Light → Pyrimidine dimers (thymine or cytosine).
-​ Chemical modifications.
Mechanism: Cut-and-Patch Process: Damaged nucleotides are excised and replaced.

Pathways
1.​ Global Genomic NER (GG-NER):
-​ Repairs bulky lesions across the entire genome.
-​ Slower pathway.
2.​ Transcription-Coupled NER (TC-NER):
 -​ Targets lesions on the template strand of actively transcribed genes.
-​ Lesion detection is signaled by stalling of RNA Polymerase II during transcription.
-​ Prioritizes repair of genes critical for cellular function.​
STEPS:
1.​ Recognition
2.​ DNA strand separation
3.​ Stabilization
4.​ Excise
5.​ Repair and ligation
DNA Repair Disorders
1.​ Xeroderma Pigmentosum (XP)
-​ Cause: Deficiency in Global Genomic Nucleotide Excision Repair (GG-NER).
-​ deficiencies in the XPC complex.
-​ Symptoms:
-​ Dry, scaly skin and abnormal pigmentation in sun-exposed areas.
-​ Extreme photosensitivity.
2.​ Cockayne Syndrome (CS)
-​ Cause: Deficiency in Transcription-Coupled NER (TC-NER).
-​ defects in CSA and CSB genes, affecting transcription-coupled repair.
-​ Symptoms:
-​ Photosensitivity without sun-induced pigmentation or increased skin cancer risk.
-​ Unique physical features:
-​ Deep sunken eyes, prominent ears, bird-like facial structure.
-​ Short stature, arrested sexual development, thin hair.
-​ Skeletal and neurological issues:
-​ Osteoporosis and several neurological symptoms.

Base Excision Repair (BER)

-​ Removes altered nucleotides caused by reactive chemicals like free radicals or reactive oxygen
species (ROS).
Types of Alterations
-​ Uracil: Formed from hydrolytic removal of the amino group from cytosine.
-​ 8-Oxoguanine (oxoG): Result of ROS damage.
-​ 3-Methyladenine: Formed by transfer of methyl groups from donors.
-​ DNA Glycosylases: Specialized enzymes that recognize and remove each specific type of altered
base.
Steps in BER
1.​ Recognition and Base Removal:
Specific DNA glycosylase excises the altered base, leaving an AP (apurinic/apyrimidinic) site.
2.​ Cleavage of DNA Backbone:
AP Endonuclease cleaves the DNA backbone at the AP site.
3.​ Sugar-Phosphate Removal:
DNA Polymerase β (phosphodiesterase activity) removes the sugar-phosphate remnant attached to the
excised base.
 4.​ Gap Filling:
DNA Polymerase β (polymerase activity) fills the gap with complementary nucleotides.
5.​ Sealing:
DNA Ligase III seals the strand to complete the repair.

Relevance to Alzheimer’s Disease (AD: Increased oxidative DNA damage in brain tissues of Alzheimer’s
patients.
-​ Deficiencies in BER due to:
-​ Limited processing of damaged bases by DNA glycosylases.
-​ Reduced DNA synthesis capacity of DNA Polymerase β.

Double-Strand Break (DSB) Repair

Cause= Ionizing Radiation: X-rays, gamma rays AND Chemicals: Chemotherapy agents, reactive
oxygen species (ROS).

Repair Mechanisms:
1.​ Non-Homologous End Joining (NHEJ):
Directly joins the broken DNA ends without a homologous template.
Less accurate but available throughout the cell cycle.

2.​ Homologous Recombination (HR):

Uses a homologous chromosome as a template for repair.
More accurate, but occurs only during the late S/G2 phase of the cell cycle.

Clinical Relevance: Radon and Lung Cancer

Mechanism:
-​ Radon: Radioactive gas formed from uranium decay in soil.
-​ Exposure to radon causes DSBs in lung cells.
-​ Unrepaired or misrepaired DSBs → increased risk of lung cancer.
Impact:
-​ 2nd leading cause of lung cancer in the U.S.
-​ Contributes to 15,000–22,000 lung cancer deaths annually.

14. Genetics and Disease

Genetic Mutation: A permanent change in the DNA sequence.

General Categories:
1.​ Single Base Pair/Point Mutations (Substitutions):
-​ Missense Mutation: Changes the amino acid.
-​ Nonsense Mutation: Creates a premature stop codon (e.g., Cystic Fibrosis).
-​ Silent Mutation: No change in the amino acid.
 -​Conservative Missense Mutation: The new amino acid has similar properties (e.g.,
hydrophobicity).
2.​ Copy Number Variations:
-​ Insertions/Deletions (Indels): May cause a frameshift if not a multiple of 3 nucleotides.
-​ Frameshift affects all codons after the mutation.
-​ Duplication: Copying a segment of DNA.
-​ Example: MYC gene (cell growth regulator) is duplicated in many cancers
(ovarian, breast, prostate, etc.).
-​ Repeat Expansion: Involves trinucleotide repeats.
-​ Example: Huntington's Disease.
3.​ Chromosomal Abnormalities:
-​ Types: Duplication, inversion, deletion, insertion, translocation, nondisjunction.
-​ Example: Down Syndrome (Trisomy 21).
-​ Caused by nondisjunction (failure of chromosome 21 to separate properly during
cell division).

When Mutations Can Occur

1.​ Hereditary/Germline Mutations:
-​ Passed from parent to child.
-​ Present in virtually all cells throughout life.
2.​ De Novo Mutations:
-​ Occur during germline development (egg/sperm) or immediately after fertilization.
-​ No family history, but the child has the mutation in all cells.
3.​ Acquired/Somatic Mutations:
-​ Develop in DNA during a person’s lifetime.
-​ Caused by environmental factors (e.g., UV radiation) or errors in DNA replication.

Review of Genetics:

Inheritance
Key Facts for Each Cell:
-​ Contains 2 meters of DNA (6 feet, 3 billion base pairs).
-​ 25,000 protein-coding genes.
-​ 46 chromosomes:
-​ 44 autosomal chromosomes (22 pairs).
-​ 2 sex chromosomes (1 pair; X and/or Y).
Key Terminology
●​ Allele: Alternative or variant forms of a gene (e.g., B vs. b).
●​ Homozygous: Two identical alleles for a gene (e.g., BB or bb).
●​ Heterozygous: Two different alleles for a gene (e.g., Bb).
●​ Dominant Allele:
○​ Always produces its trait when inherited (e.g., B).
●​ Recessive Allele:
○​ Produces its trait only when homozygous (e.g., b).
Example: Eye Color
-​ B: Brown (dominant allele).
-​ b: Blue (recessive allele).
-​ Genotype: Bb (heterozygous).
-​ Produces brown eyes (due to dominance of B).

Single Gene Disorders: Disorders caused by abnormal or missing genes.

1.​ Autosomal Dominant
2.​ Autosomal Recessive
3.​ Sex-linked Inheritance

1.​ Autosomal Dominant

Key Features:
-​ Inheritance of a dominant disease-causing allele.
-​ 50% chance of being affected if one parent carries the allele.
-​ Appears in every generation; males and females equally affected.
Example: Huntington’s Disease (HD):

-​ Cause: Expansion of CAG repeats in the HTT gene.

-​ HTT encodes the huntingtin protein (important in neurons).
-​ Symptoms:
-​ Personality changes: Depression, irritability, anxiety, obsessive behavior.
-​ Cognitive decline: Loss of focus, planning, memory, and decision-making.
-​ Physical deterioration: Weight loss, involuntary movements, loss of coordination.
-​ Progression: Leads to complete incapacitation and death; onset typically between
30–45 years.
-​ Inheritance:
-​ Each child of an affected parent has a 50% chance of inheriting.
-​ ~1 in 7,000 Canadians affected.

2.​ Autosomal Recessive

Key Features:
-​ Disease manifests when an individual inherits two defective alleles (homozygous).
-​ Parents are carriers but do not show symptoms.
-​ 25% chance for a child to be affected.\
-​ More common in closely related parents.
-​ Affects males and females equally.
Example: Cystic Fibrosis (CF):
-​ Cause: Mutation in the CFTR gene (regulates sweat, digestive fluids, and mucus).
-​ Symptoms:
-​ Breathing difficulties, chronic lung infections, permanent lung damage.
-​ Difficulty digesting food, failure to grow.
Statistics:
 -​ Affects 1 in 3,600 children in Canada.
-​ 1 in 25 Canadians is a carrier

3.​ Sex-linked Inheritance

Key Features:
-​ Caused by a defective gene on the X chromosome.
-​ In males:
-​ Defective X is unmasked (no second X to compensate), and the trait is expressed.
-​ In females:
-​ Heterozygous females are carriers but typically not affected.
-​ Male carriers transmit the defective X to daughters, never to sons.
Example: Color Blindness:
-​ Cause: Mutations in genes producing photopigments (on X chromosome).
-​ Symptoms: Inability or reduced ability to perceive colors (e.g., red/green discrimination).
-​ Statistics:
-​ 1 in 10 males affected by some form of color blindness.
-​ Rare in females (second X compensates).

Multi-Gene Disorders
Definition: Disorders influenced by multiple genes and environmental factors.
Familial Inheritance:
-​ Patterns: Diseases tend to run in families, but inheritance is not completely understood.
-​ Mechanism: Several genes interact, often influenced by environmental factors.
Examples:
-​ Diabetes
-​ Allergies

KAROTYPES: Characterization of chromosomes by shape, size, and number.

Purposes of Karyotyping
-​ Determine if parental chromosomes have abnormalities that can be inherited.
-​ Identify chromosome defects preventing pregnancy.
-​ Detect chromosome defects in a fetus.
-​ Investigate chromosome defects causing birth defects in a baby.
-​ Determine the sex of a person (presence of the Y chromosome).
Process of Karyotyping
1.​ Biological Sample: Blood, amniotic fluid, or placenta.
2.​ Mitogen: Stimulates cells to divide.
3.​ Colchicine: Arrests cells in metaphase by poisoning the mitotic spindle.
4.​ Cell Preparation:
-​ Nuclei Swelling: Burst cells to release chromosomes.
-​ Trypsin Treatment: Degrades proteins (e.g., histones) bound to DNA, loosening
chromosome structure.
5.​ Giemsa Staining:
 -​ Produces G-banding: Allows precise identification of each chromosome's
structure and potential abnormalities.
-​ Light bands: Regions with fewer A-T bases.
-​ Dark bands: Regions with more A-T bases.
-​ Each chromosome is identified by its unique banding pattern.

CHROMOSMAL ABNORMALITIES: Missing, extra, or irregular portions of chromosomal DNA,

leading to problems in growth, development, and bodily functions.
1.​ Numerical anomalies → aneuploidy (i.e. monosomy, trisomy)
-​ Aneuploidy
-​ Presence/absence of 1 or more chromosomes
2.​ structural anomalies → deletions, duplications, translocations, inversions, insertions

Types of Chromosomal Abnormalities:

1.​ Gamete Nondisjunction
-​ In sex cell during meiosis I or II
-​ Autosomal chromosomes
-​ Sex chromosomes
2.​ Zygote Nondisjunction
-​ After fertilization
3.​ Chromosome deletions
4.​ Chromosome duplications
5.​ Chromosome translocations

1. Gamete Nondisjunction

A. Gamete Nondisjunction (Autosomal Chromosomes)

●​ Definition: Failure of homologous chromosomes to separate during meiosis I or II.

●​ Mechanism: Caused by spindle structure disturbances during anaphase I/II.
●​ Impacts:
1.​ Affected germ cells may have an extra or missing chromosome.
2.​ Following fertilization, the embryo may result in:
■​ Trisomy (e.g., Trisomy 21).
■​ Monosomy (generally not viable).
●​ Examples:
1.​ Trisomy 13 (Patau Syndrome):
■​ Symptoms: Cleft palate, severe intellectual disability, seizures, skeletal
abnormalities, microcephaly, congenital heart defects.
■​ Prognosis: >80% die in the first year.
2.​ Trisomy 18 (Edwards Syndrome):
■​ Symptoms: Low birth weight, mental delay, microcephaly, heart/kidney defects.
■​ Prognosis: >50% die in the first week.
 3.​ Trisomy 21 (Down Syndrome):
■​ Symptoms: Distinctive facial features, developmental/social delays, congenital
heart anomalies.
■​ Prognosis: Can live independent, productive lives into adulthood.
■​ Incidence increases with maternal age.

B. Gamete Nondisjunction (Sex Chromosomes)

●​ Definition: Failure to separate X/Y chromosomes during meiosis.

●​ Impacts:
1.​ Less severe phenotype due to:
■​ X-inactivation in females.
■​ Low gene content on Y chromosome.
2.​ Symptoms: Puberty delay, amenorrhea (absence of menstruation), infertility, ambiguous
genitalia.
●​ Examples:
1.​ Klinefelter’s Syndrome (XXY):
■​ External genitalia = male, but testicles are atrophic, sterile.
■​ Feminized body configuration, possible breast hypertrophy.
■​ Normal intelligence (may decrease with >2 X chromosomes).
■​ Incidence: ~1/500–1,000 males in Canada.
2.​ Turner’s Syndrome (X0):
■​ External genitalia = female, sterile (underdeveloped uterus/ovaries).
■​ Secondary sex characteristics underdeveloped, short stature, cardiovascular
malformations.
■​ Incidence: ~1/2,000 females in Canada.
3.​ Triple X Syndrome (XXX):
■​ Subtle phenotype: Tall stature, normal fertility/sexual development, possible
learning disabilities or motor coordination issues.
■​ Associated with advanced maternal age.
■​ Incidence: ~1/1,400 females in Canada.
4.​ XYY Syndrome (Super-Male):
■​ Normal phenotype, some may have increased growth velocity, severe acne, or
learning disabilities.
■​ Normal sexual development and fertility.
■​ Associated with advanced maternal age.
■​ Incidence: ~1/1,000 males in Canada.

2. Zygote nondisjunction (mitotic nondisjunction)

Definition: Occurs in somatic cells when sister chromatids fail to separate during mitosis.

Mechanism:
-​ Normal haploid gametes → normal diploid zygote.
-​ Error during anaphase leads to populations of cells with different karyotypes (mosaicism) as the
zygote undergoes subsequent mitotic divisions.
Examples:
-​ Down Syndrome
-​ Turner’s Syndrome
-​ Triple X Syndrome

3. Chromosome deletions
Definition: A segment of a chromosome is deleted during meiosis, often during crossover.

Features:
-​ Can occur on any chromosome, at any allele, and vary in size.
-​ Impact depends on the location and genes missing.
-​ Significant deletions are usually incompatible with fetal survival.
Example: Cri Du Chat Syndrome:
-​ Cause: Deletion on the short arm of chromosome 5.
-​ Symptoms:
-​ High-pitched “cat-like” cry (larynx defect).
-​ Intellectual disability, delayed development, microcephaly, hypotonia (reduced strength).
-​ Distinctive facial features: widely set eyes, low-set ears, small jaw, rounded face.
-​ Some cases involve heart defects.
-​ Can result in early death.

4. Chromosome duplications
Definition: A part of a chromosome is duplicated during meiosis.

Features:
-​ Results in partial trisomy (three copies of the duplicated region).
-​ Gene dosage effect: Phenotype results from altered levels of gene products.
-​ Types:
-​ Tandem duplication: Duplicated section is adjacent to the original.
-​ Displaced duplication: Duplicated sections are separated by non-duplicated regions.
-​ Typically less severe than deletions.

Example: Charcot-Marie-Tooth Disease (CMT):

-​ Cause: Duplication of a large region on the short arm of chromosome 17.
-​ Effects:
-​ Overexpression of PMP22 (myelin protein).
-​ Abnormal myelin production → nerve damage (demyelinating neuropathy).
-​ Symptoms:
-​ Progressive loss of muscle tissue and touch sensation.
-​ Breathing, hearing, and vision may also be affected.
-​ Onset in late childhood/early adulthood.
-​ Varies in severity and progression.

5 Chromosome Translocations
Definition: A piece of one chromosome attaches to another.

Types:
-​ Balanced Translocation (in somatic cells):
-​ No net gain or loss of genetic material → minimal effect on function.
-​ May create gene fusion proteins, increasing malignancy risk.
-​ Example: Breakage during transcription can trigger abnormal fusion.
-​ Unbalanced Translocation (in gametes):
-​ Results in gain or loss of genetic material.
-​ Causes miscarriages or severe birth defects.

Example: Chronic Myelogenous Leukemia (CML):

-​ Cause: Philadelphia Chromosome:
-​ Part of chromosome 22 translocated to chromosome 9.
-​ Fusion of ABL gene (Ch9) with BCR gene (Ch22).
-​ Effects:
-​ Novel fusion protein (kinase) promotes cell proliferation but is improperly regulated.
-​ Leads to malignancy.

Importance of Early Diagnosis

Key Benefits:
-​ Enables detection and treatment of genetic diseases.
-​ In some cases, allows for prevention.
-​ Example: Phenylketonuria (PKU):
-​ Autosomal recessive disorder causing phenylalanine build-up in the body.
-​ Managed through dietary adjustments.

Diagnosis Methods

A. Pre-natal Diagnosis:
1.​ Amniocentesis:
-​ Process: Small amount of amniotic fluid withdrawn (14th–18th week of pregnancy).
-​ Can detect ~200 genetic diseases.
2.​ Chorionic Villous Sampling (CVS):
-​ Process: Removal of chorionic villi cells from the placenta (8th–10th week of
pregnancy).
-​ Advantage: Provides results earlier than amniocentesis.

B. Post-natal Diagnosis:
1.​ Newborn Blood Sampling:
-​ Purpose: Identifies 28 conditions, including:
-​ Metabolic disorders
-​ Endocrine disorders
-​ Blood disorders
 -​ Cystic fibrosis
Gene Therapy: Insertion, alteration, or removal of a gene in an individual's cells/tissues to treat disease
(genetic engineering).
Limitations:
-​ Longevity of new gene integration.
-​ Challenges with multiple copies of gene insertion.
-​ Immune response to viral vectors.
-​ Ineffectiveness for multi-gene disorders.
-​ Risk of mutagenesis (gene mutations during insertion).
-​ Long-term outcomes remain unclear.

15. Cell Signalling and G-Coupled Receptors

Cell Signaling Overview
-​ Purpose: Enables cells to respond to external stimuli for survival.
-​ Mechanism:
-​ Signal molecules (ligands) interact with cell-surface or intracellular receptors.
-​ Receptors initiate intracellular actions via a cascade of protein conformational changes.

Types of Signaling
Autocrine Signaling:
-​ Cell responds to its own secreted messengers.
-​ Self-regulation (induce/suppress own activity).
Paracrine Signaling:
-​ Messenger molecules act on nearby cells.
-​ Localized regulation between adjacent cells.
Endocrine Signaling:
-​ Hormones travel via bloodstream to distant target cells.
-​ Systemic communication.

Signal Transduction

1.​ Key Components:

-​ Ligands: Signal molecules (e.g., steroids, neurotransmitters, insulin).
-​ Receptors: Proteins on target cells that bind ligands and undergo conformational changes.
2.​ Transmission Routes:
-​ Second Messenger Generation:
-​ Effector enzymes produce intracellular second messengers.
-​ Second messengers modulate target protein activity.
-​ Protein Recruitment Cascade:
-​ Intracellular signaling proteins recruited to receptor domains.
-​ Protein activation cascade initiated.

Effects of Protein Phosphorylation

 -​ Alters protein behavior through:
-​ Enzyme activation/inactivation.
-​ Changes in protein-protein interactions or subcellular location.
-​ Triggering protein degradation.
-​ Phosphorylation patterns vary by cell type (e.g., for treatment decisions in cancer).
-​ Phosphorylation Is a Central Mechanism for Circadian Control of Metabolism and Physiology

Signaling Cascades

●​ Mechanism:
○​ Proteins in a cascade alter each other’s conformation (via phosphorylation).
○​ Enzymes involved:
■​ Kinases: Add phosphate groups (+Pi).
■​ Phosphatases: Remove phosphate groups (-Pi).
○​ Target amino acids: Serine, threonine, tyrosine.
●​ Outcomes:
○​ Phosphorylation of transcription factors → gene transcription.
○​ Regulation of:
■​ Metabolic enzyme activity.
■​ Cell mobility.
■​ DNA synthesis and replication.
■​ Cell death.

Types of Receptors
1.​ G Protein-Coupled Receptors (GPCRs):
-​ Largest family of membrane proteins.
-​ 7 transmembrane helical domains.
-​ Coupled to cytoplasmic G proteins (α, β, γ subunits).
-​ Use GTP to activate effector proteins and generate second messengers.
2.​ Receptor Protein-Tyrosine Kinases (RTKs):
-​ Key in growth and differentiation signaling pathways.
3.​ Ligand-Gated Channels:
-​ Allow ion passage upon ligand binding.
4.​ Steroid Hormone Receptors:
-​ Nuclear receptors that interact directly with DNA to regulate gene expression.
5.​ Specialized Receptors:
-​ Include B-cell and T-cell receptors for immune responses.

G Protein-Coupled Receptors (GPCRs): Signal Transduction Process

1.​ Ligand Binding:
Ligand binds to the extracellular domain of GPCR.
Induces receptor conformational change, increasing affinity for the G protein.
2.​ G Protein Activation:
Gα exchanges GDP for GTP, activating the G protein.
Multiple G proteins can be activated.
3.​ Effector Interaction:
GTP-Gα dissociates from Gβγ subunits.
GTP-Gα binds to an effector protein, activating/inhibiting it.
4.​ Effector Proteins and Second Messengers:
Adenylyl Cyclase: Converts ATP to cAMP.
Phospholipase C: Generates IP3 and DAG.
cGMP Phosphodiesterase: Modulates cGMP levels.
5.​ GTP Hydrolysis (Self-Turn Off):
Gα hydrolyzes GTP to GDP, inactivating itself.
Conformational change increases affinity for Gβγ and decreases affinity for effectors.
6.​ Receptor Desensitization:
GPCR is phosphorylated by G protein-coupled receptor kinase (GRK).
Arrestin binds to the receptor, preventing G protein binding and promoting:
Internalization of GPCR.
Signaling from internalized receptors.
Degradation or recycling to the membrane.

Regulation of GPCR Signaling

-​ Signal Strength and Duration:
-​ Determined by the rate of GTP hydrolysis by Gα.
-​ Gα has weak GTPase activity, slowly hydrolyzing GTP.
-​ Regulators of G Protein Signaling (RGSs):
-​ Accelerate GTP hydrolysis to terminate the signal.

G Protein-Coupled Receptors (GPCRs): Effectors and Second Messengers

1.​ Gs (Stimulatory G Protein):

Effector: Activates Adenylyl Cyclase.
Second Messenger: Increases cAMP levels.
2.​ Gi (Inhibitory G Protein):
Effector: Inhibits Adenylyl Cyclase.
Second Messenger: Decreases cAMP levels.
3.​ Gq:
Effector: Activates Phospholipase C-beta (PLCβ).
Second Messengers:
-​ IP3 (Inositol Triphosphate): Mobilizes calcium from
intracellular stores.
-​ DAG (Diacylglycerol): Activates Protein Kinase C (PKC).
4.​ G12/13:
Effector: Activates small G proteins.
Impact: Excessive activation can lead to:
-​ Cell proliferation + Malignancies (cancer development).
SECOND MESSENGERS: Molecules released into the cytoplasm following ligand binding to a
receptor.
Role:
-​ Amplify the cellular response to a single extracellular ligand.
-​ Many second messengers are produced by a single effector enzyme.
Common Second Messenger Systems:
-​ Cyclic Nucleotides: cAMP and cGMP.
-​ Phosphatidylinositol Derivatives: Inositol trisphosphate (IP3) and Diacylglycerol (DAG).
-​ Calcium Ions (Ca²⁺).

1.​ cAMP (Cyclic Adenosine Monophosphate)

●​ Synthesis:
○​ Produced by Adenylyl Cyclase from ATP.
○​ Stimulated by Gs subunits and inhibited by Gi subunits.
●​ Properties:
○​ Capable of diffusing to different sites within the cell.
○​ Activates various cellular activities.

Functions of cAMP:

1.​ Activates Protein Kinases:

○​ Leads to phosphorylation of proteins involved in:
■​ Cytoskeletal dynamics.
■​ Protein synthesis.
■​ Glycogen metabolism (breakdown and formation).
■​ Fatty acid formation.
■​ Activation of calcium channels.
2.​ Regulation via Protein Kinase A (PKA):
○​ Structure:
■​ Holoenzyme: Active enzyme formed from enzyme-coenzyme interaction.
■​ Subunits:
■​ Regulatory (R): Binds cAMP.
■​ Catalytic (C): Free upon high cAMP levels to phosphorylate proteins.
○​ Activation Process:
■​ Low cAMP → PKA inactive.
■​ High cAMP → cAMP binds R subunits, releasing active C subunits.
○​ Function:
■​ Phosphorylates over 100 substrates.
■​ Regulates various cell activities via protein modification.
3.​ Feedback Regulation:
○​ cAMP is broken down by Phosphodiesterase (PDE).
○​ Phosphorylated PDE forms a feedback loop to terminate cAMP signaling.
Protein Kinase A Anchoring Proteins (AKAPs)
-​ Serve as signaling hubs by scaffolding interactions.
-​ Localize PKA to specific cellular regions.
-​ Over 50 known AKAPs coordinate precise cellular signaling pathways.

Adrenergic Receptors and Blood Glucose Regulation

Definition: A class of GPCRs that respond to catecholamines like norepinephrine and epinephrine.

Characteristics:
-​ Nine receptor types, grouped into α- and β-receptors.
-​ Mediate the fight-or-flight response by activating different signaling pathways in various tissues.
Examples of Adrenergic Receptors:
1.​ β-Adrenergic Receptor (Cardiac Muscle Cells):
-​ Mechanism:
-​ Activates Gs subunit → increases cAMP → enhances cardiac contraction rate
and force.
-​ Function: Boosts heart performance during stress.
2.​ α-Adrenergic Receptor (Intestinal Smooth Muscle Cells):

-​ Mechanism:
-​ Activates Gi subunit → inhibits cAMP → leads to muscle relaxation.
-​ Function: Redirects energy by relaxing intestinal muscles during stress.

Regulation of Blood Glucose via GPCRs and RTKs

1.​ GPCR Pathways (cAMP-Mediated):

Glucagon:
-​ Endocrine hormone released by the pancreas in
response to low glucose levels.
-​ Binds to glucagon GPCR in liver cells.
-​ Stimulates glycogen breakdown → releases glucose
into the bloodstream.
Epinephrine:
-​ Endocrine hormone from adrenal glands during the
fight-or-flight response.
-​ Binds to β-Adrenergic receptors in liver cells.
-​ Promotes glycogen breakdown → releases glucose.

2.​ RTK Pathways (Receptor Protein-Tyrosine Kinases):

Insulin:
-​ Released from the pancreas in response to high glucose levels.
-​ Binds to insulin RTK.
-​ Stimulates glucose uptake and storage as glycogen.
 ★​ CREB: cAMP Response Element Binding Protein
-​ Role:
-​ Activated by cAMP.
-​ Regulates transcription of genes involved in glucose metabolism and other
pathways.

G Proteins and Human Disease

Bacterial Toxins:
-​ G proteins are critical for cellular function, making them targets for pathogens.
-​ Example: Cholera Toxin (Vibrio cholerae):
-​ Modifies the Gs subunit, preventing GTPase activity.
-​ Causes prolonged Adenylyl Cyclase activation → excessive cAMP production.
-​ Effects:
-​ High cAMP leads to loss of salts and water from intestinal epithelia.
-​ Symptoms: Severe watery diarrhea and dehydration.
-​ Pathogen Benefit: Enhances transmission by contaminating
water/environment to spread to new hosts.
McCune-Albright Syndrome
-​ Cause: Mutation in the GNAS1 gene (located on Chromosome 20).
-​ Gene Function: GNAS1 encodes the Gαs subunit of G proteins.
-​ Mutation Effect:
-​ Compromised GTPase activity of Gαs.
-​ Constitutive activation of cAMP-driven pathways without hormone stimulation.

Signalling molecules that rely on GPCRs + Effects of Activating Gαs Mutations:

MSH - melanocyte-stimulating hormones + Melanin - pigmentation
LH – luteinizing hormone + Estrogen (LH increases production of estrogen by ovary) – puberty
GNRH - Gonadotropin-releasing hormone + Growth Hormone - gigantism
TSH – thyroid stimulating hormone + Thyroid hormones – abnormal bone turnover and hypothyroidism

16. Signal Transduction: IP3, DAG and PKC

Major Routes of Signal Transmission
1.​ Second Messenger Generation:
-​ Signal activates an effector enzyme.
-​ Effector produces intracellular second messengers (e.g., cAMP, IP3).
Second messengers:
-​ Activate or inactivate target proteins.
2.​ Protein Recruitment Cascades:
-​ Signal recruits signaling proteins to receptor domains.
-​ Triggers a protein activation cascade.

Light-Triggered Signaling in Rod Cells

Key Components
1.​ Receptor: Rhodopsin
-​ Light-sensitive GPCR in rod cells of the retina.
2.​ G Protein: Transducin (Gαt).
3.​ Effector Enzyme: cGMP Phosphodiesterase (PDE).
4.​ Second Messenger: cGMP.

Mechanism of Light-Triggered Signaling

1.​ In the Dark:
●​ cGMP levels are high.
●​ cGMP-dependent Na⁺ and Ca²⁺ ion channels are
open.
●​ Membrane is in a depolarized state.
2.​ When Light Activates Rhodopsin:
●​ Photon activates GPCR (Rhodopsin).
●​ Rhodopsin activates the G-protein transducin
(Gαt).
●​ Transducin activates cGMP Phosphodiesterase
(PDE).
●​ cGMP → GMP, reducing cytosolic cGMP levels.
●​ Ion channels close, causing:
○​ Membrane hyperpolarization (more negative membrane potential).
○​ Signal sent to the visual center of the brain.
Bradyopsia (Slow Vision)
Cause: Mutations in RGS9, which normally inactivates transducin to restore cGMP levels during light
termination.
Effect:
-​ Slower cGMP increase → Na⁺ and Ca²⁺ channels reopen more slowly.
-​ Delayed membrane depolarization (return to resting state).
-​ Difficulty adjusting to changes in light, e.g., temporary blindness in bright light.

★​ Phospholipids in cell membrane can be targeted for

cleavage by phospholipase enzymes → cleave at
specific sites
★​ By-products of cleavage can act as second messengers
within the cytoplasm (IP3 and DAG can act as second
messengers within the cytoplasm)

Second Messengers – IP3 & DAG:

Key Components
1.​ G Protein: Gαq.
2.​ Effector Enzyme: Phospholipase C (PLC).
3.​ Second Messengers:
-​ Inositol trisphosphate (IP3).
 -​ Diacylglycerol (DAG).
4.​ Receptors: many diff ones

STEPS:
1.​ Ligand binds receptor → Gαq activated.
2.​ Gαq activates PLCβ.
3.​ PLCβ cleaves PIP2 → IP3 + DAG.
4.​ DAG activates PKC.
5.​ IP3 triggers Ca²⁺ release, amplifying cellular responses.

Actions of Second Messengers

1.​ DAG:
-​ Recruits Protein Kinase C (PKC) to the membrane.
-​ PKC Activation:
-​ Regulates:
-​ Cellular growth and differentiation.
-​ Cellular metabolism.
-​ Cell death.
2.​ IP3:
-​ Diffuses into the cytosol and binds to calcium channels on the smooth endoplasmic
reticulum (SER).
-​ Ca²⁺ Release → most widely used second messenger:
-​ Increases cytosolic calcium, leading to:
-​ Recruitment of PKC to DAG (events mediated by PKC via DAG pathways.
-​ Cellular effects such as:
-​ Muscle contraction.
-​ Release of histamine.

★​ DAG → PKC signalling = causes diverse responses: serotonin, histamine release, etc.
★​ IP3 → Ca2+ signalling = causes diverse response: contractions

★​ Abnormal PKC signalling in several diseases • CVD, stroke, diabetes, asthma,

autoimmunity/inflammation, neurological diseases

Cytosolic Calcium Regulation

Resting Levels: Maintained at ~0.1 mM (low) to optimize the signal-to-noise ratio.

Mechanisms to Maintain Low Ca²⁺ Levels:

-​ Active Transport:
-​ Removes Ca²⁺ from the cytosol out of the cell.
-​ Sequestration:
-​ Stores Ca²⁺ in the endoplasmic reticulum (ER).
-​ Calcium Influx and Release
Calcium Influx and Release
1.​ Calcium Entry:
-​ Occurs via voltage-activated Ca²⁺ channels in response to changes in membrane potential.
2.​ Calcium-Induced Calcium Release (CICR):
-​ Involves ryanodine receptors (RyRs) and IP₃ receptors (IP₃Rs) on the smooth ER.
-​ Activation:
-​ RyRs: Activated directly by Ca²⁺ binding.
-​ IP₃Rs: Activated by IP₃ binding, with Ca²⁺ modulating their sensitivity.
-​ Function:
-​ RyRs: Trigger rapid and substantial Ca²⁺ release (e.g., for muscle contraction).
-​ IP₃Rs: Release Ca²⁺ in response to extracellular signals, influencing various
cellular processes.
EX: Calcium Signaling in Fertilization: Sperm binding activates GPCR → triggers IP₃-mediated Ca²⁺
release. Creates a calcium wave dependent on CICR.

17. Signal Transduction: RTKs and

MAPKs
G- COUPLE PATHWAYS →

Receptor Protein-Tyrosine Kinases (RTKs)

Structure: Transmembrane receptors spanning the plasma
membrane once. Intracellular domain has tyrosine kinase
activity.
Common Ligands:
Insulin, VEGF, PDGF, EGF, FGF.
Function: Mediate various effects, such as growth, differentiation, and repair (e.g., skin regeneration).

STEPS:
1.​ Ligand Binding → Receptor Dimerization:
Two modes of dimerization:
a)​ Ligand-Mediated Dimerization:
-​ One ligand binds to both receptor proteins simultaneously (e.g., PDGF).
b)​ Receptor-Mediated Dimerization:
-​ Two ligands bind separately to two receptor proteins (e.g., EGF).
2.​ Trans-Autophosphorylation:
-​ Receptors phosphorylate tyrosine residues on their intracellular domains.
-​ Creates binding sites for downstream signaling molecules.
3.​ Relay Protein Activation:
-​ Inactive relay proteins are recruited to the receptor.
-​ Proteins are phosphorylated, becoming active, and initiate a cellular response.
-​ Interaction is mediated by specialized domains in relay proteins:
-​ SH2 (Src Homology 2).
-​ PTB (Phosphotyrosine-Binding).
Ending the Response
Importance:
-​ Cells must stop responding to signals to prevent overactivation.
-​ Failure to terminate growth factor signaling can lead to uncontrolled mitosis and cancer.

Relay Proteins
-​ RTKs recruit and activate relay proteins, leading to diverse cellular responses.
Types of Relay Proteins:
1.​ Adaptor Proteins:
-​ Function: Link signaling proteins (e.g., Grb2).
2.​ Docking Proteins:
-​ Function: Provide docking sites for multiple signaling proteins (e.g., IRS in insulin
signaling).
3.​ Transcription Factors:
-​ Function: Translocate to the nucleus upon activation and alter gene transcription (e.g.,
STAT).
4.​ Enzymes:
-​ Function: Perform signaling functions (e.g., PI3K, PLCγ, shp2).
Multiple Relay Proteins: Allow different signaling pathways to trigger varied cellular responses.

Three Major RTK Signaling Pathways

1. PLCγ → IP3/DAG Pathway:

●​ Mechanism:
○​ PLCγ binds to RTK via SH2 domain.
○​ Tyrosine phosphorylation activates PLCγ.
○​ PLCγ cleaves PIP2 into IP3 and DAG.
●​ Outcomes:
○​ IP3 → Releases Ca²⁺ from the endoplasmic reticulum.
○​ DAG → Activates Protein Kinase C (PKC).

2. PIP3 → AKT Pathway:

●​ Mechanism:
○​ RTK phosphorylates IRS (Insulin Receptor Substrate) docking protein.
○​ PI3K is recruited and phosphorylates PIP2 → PIP3.
○​ PIP3 recruits PDK1, PDK2, and Akt to the membrane.
○​ Akt is phosphorylated at Thr308 and Ser473, activating it.
●​ Outcomes:
○​ Regulates metabolism, cell survival, and growth.

3. Ras → MAPK Pathway:

●​ Mechanism:
 ○​ Stimulus activates Ras, initiating a cascade:
■​ MAP3K (e.g., Raf) → MAP2K (e.g., MEK) → MAPK (e.g.,
ERK).
●​ Outcomes:
○​ Regulates cell proliferation, differentiation, gene expression, survival,
and apoptosis.
●​ Three MAPK Pathways in Mammals:
○​ ERK, p38, JNK.

Summary
RTKs activate multiple relay proteins, leading to distinct pathways:
1.​ PLCγ → IP3/DAG: Calcium signaling and PKC activation.
2.​ PIP3 → AKT: Cell growth, survival, and metabolism.
3.​ Ras → MAPK: Proliferation, differentiation, and stress responses.
Significance: These pathways regulate essential cellular processes, including growth, repair, and survival.

Convergence: Multiple receptors → one effector.

-​ Definition: Signals from different receptors, each binding its own ligand, converge to activate a
common effector.
Divergence: One receptor → multiple effectors.
-​ Definition: Signals from the same receptor-ligand complex activate multiple effectors,
triggering varied cellular responses.
Cross-Talk: Interaction between pathways → modulation of signaling outcomes.
-​ Definition: Signals are shared between different pathways, allowing them to enhance or repress
each other.