DNA Transcription

RNA Structure and Types

• RNA is rapidly degraded

• RNA is mainly in single-stranded form.
• It contains uracil instead of thymine
• It contains ribose instead of deoxyribose

Primary and Secondary RNA Structure

In the secondary structure, RNA contains GC and AU hydrogen bonds

Types of RNA
Prokaryote and Eukaryote:
- Messenger RNA = mRNA
- Ribosomal RNA = rRNA
- Transporting RNA = tRNA
Eukaryote:
- Small nuclear RNA = snRNA
- Small nucleolar RNA = snoRNA
- Small cytoplasmic RNA = scRNA
- Micro RNA = miRNA
- Small interfering RNA = siRNA
- Piwi-interacting RNA = piRNA
- Long non-coding RNA = lncRNA

Cell location and function of different types of RNA molecules

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In eukaryotic cells, non-coding protein RNA of human transcriptome makes up the
majority

Transcriptome = is a set of RNA molecules in a cell

The majority of RNA in the transcriptome is non-protein coding RNA

Eukaryotic mRNA is formed during processing pre-mRNA in the nucleus

Pre-mRNA needs to be protected from enzymes that can degrade and destroy it. It is
protected by:
- A nucleotide 5' cap (attachment of 7-methylguanosine at the 5' end of pre-mRNA)
- A poly-A tail at the 3' end of the pre-mRNA

It is still not ready for making proteins. The pre-mRNA has areas of introns and exons.
Exons = coding sequences which can be used for protein translation
Introns = non-coding sequences not used for protein translation. It must be removed.

Splicing removes introns and joins exons

mRNA carries information about proteins, copied from the newly synthesized DNA
strand
Eukaryotic mRNA makes up about 5% of total RNA.
The coding region contains codon sequence
information on the amino acid sequence in the
protein

The 3' UTR region of the mRNA in eukaryotic

cells contains information about its degradation

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Prokaryotic mRNA is polycistronic - is an mRNA that encodes several proteins
It does not possess a nucleotide cap and poly-A tail.
- Prokaryotic mRNA is not formed from pre-mRNA
- It is the primary transcript for genes encoding proteins
- Prokaryotic mRNA contains Shine-Dalgarno (SD) sequence

Shine-Dalgarno (SD) sequence = is a ribosomal binding site (with 16S rRNA) in bacterial
mRNA, generally located around eight bases upstream of the start codon AUG.
- The sequence is base-paired to a complementary sequence on the 16S rRNA.
- It initiates protein synthesis by aligning the ribosome with the start codon - AUG

tRNA (transfer RNA) - incorporate free amino acids from the cytoplasm into a
polypeptide chain.

There are three folds in the cloverleaf tRNA. The tRNA molecules are made up of 73 to
93 ribonucleotides. The acceptor's arm carries an amino acid. It has four arms namely:
○ Anticodon arm - has three anticodon nucleotides, which will join with the
complementary codon in mRNA during protein synthesis i.e. three nucleotides
in the tRNA pairs with three nucleotides of mRNA.
○ D arm - DHU (dihydrouridine), contains information on which amino acid
may be attached to the tRNA
○ Variable arm
○ T C arm - ribothymidine is a modified base called pseudouridine. Used to
connect to the ribosome
○ Acceptor arm
The amino acid acceptor and the anticodon arms are oriented in opposite directions.

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Structure of Ribosomes in Prokaryotic Cells
Ribosome = protein complex with nucleic acids used for peptide bond
formation in translation (production of peptides)
- Ribosome = rRNA + proteins
- rRNA accounts for approx. 80% of total RNA in eukaryotic cells

Eukaryotic cells contain a small amount of small nuclear RNA (snRNA)

snRNA = non-coding RNA acts as a ribozyme involved in intron excision
- It is also called UsnRNA due to the high content of uridine nucleotides.
- Essential for removing introns from pre-mRNA to mRNA
- Small nuclear ribonucleoproteins (snRNP) = 8 Sm proteins + 1 snRNA
○ snRNP - an essential component of the spliceosome involved in
intron excision

Gene Transcription = the process of RNA synthesis on a DNA template by

various RNA polymerases - transcription of information from DNA into RNA.
Template is read 3' to 5' direction, and a new RNA molecule is formed in the
5' - 3' direction.

All known RNA polymerases require:

- Template, a dsDNA
- All four precursors: ATP, GTP, UTP, CTP
- Mg 2+ or Mn 2+ ions
The RNA strand is synthesized in the 5' - 3' direction.

They do not need a starter

They do not have exonuclease activity 3' - 5' (proofreading)

The function of RNA polymerase in Transcription

- Search for transcription initiation sites = promotors in DNA
- Unwind a short dsDNA fragment to create an ssDNA template
- Choose the correct triphosphate of ribonucleoside and catalyse the
formation of a phosphodiester bond
- Detect the transcription terminal signal
- Interact with protein activators and repressors that modulate the
frequency of transcription initiation

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DNA Transcription
Gene transcription includes initiation, elongation, and termination
→ Transcription initiation
RNA polymerase binds to the gene promoter, which disassembles the DNA double helix throughout several
base pairs.
→ Transcription elongation
The movement of RNA polymerase along the DNA molecule is accompanied by the helix unwinding and
attachment of nucleotides complementary to the DNA template strand sequence.
→ Transcription termination
Termination of transcription after reaching the termination region or Rho protein, dissociation of transcript
and polymerase from template DNA.

Gene Regulation
⚫ The regulation of gene expression in prokaryotic cells occurs at the transcriptional level.
⚫ There are two majors kinds of proteins that control prokaryotic transcription: repressors and activators.
○ Repressors bind to an operator region to block the action of RNA polymerase.
○ Activators bind to the promoter to enhance the binding of RNA polymerase.
⚫ Inducer molecules can increase transcription either by inactivating repressors or by activating activator
proteins.

The lac operon is an example of positive regulation genes Transcription by the

substrate. The lac operon (lactose operon) is required for the transport and metabolism
of lactose in Escherichia coli and many other enteric bacteria.

- The lac operon is activated by the CAP (catabolite activator protein), which binds to the promoter to
stabilize RNA polymerase binding.
- CAP is itself activated by cAMP, whose concentration rises as the concentration of glucose falls.
- However, the lac operon also requires the presence of lactose for transcription to occur.
- Lactose inactivates the lac repressor, and prevents the repressor protein from binding to the lac
operator. With the repressor inactivated, transcription may proceed.
- Therefore glucose must be absent and lactose must be present for effective transcription of the lac
operon.

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The trp Operon
The tryptophan operon is an example of negative regulation genes
transcription by the substrate.
The Tryptophan operon is a group of genes used or transcribed, together with
that codes for the enzymes for the synthesis of tryptophan.
Within the operon's regulatory sequence, the operator is bound to the repressor protein in the presence of
tryptophan preventing transcription.

Cis-Regulatory Elements (CREs) in Bacterial DNA = regions of non-coding DNA that regulate the
transcription of neighbouring genes
Operators are CREs in prokaryotes and some eukaryotes, where activators and repressors bind

Histone and DNA Modifying Enzymes are involved in reversibly rebuilding heterochromatin into
euchromatin.
- HAT = histone acetyltransferase
- HDAC = histone deacetylase
- HMT = histone methyltransferase
- DNA methyltransferase
Acetylation = (block positive NH2-K) reduces the interaction of histones with negatively charged DNA
Deacetylation = (open positive e-NH2-K) increases the interaction of histones with negatively charged DNA

Histone acetylation is associated with higher transcriptional activity

DNA methylation converts C in CpG dinucleotide into 5-methyl-C which promotes the conversion of
euchromatin into heterochromatin and stops transcription.
- It attracts HDAC - removes the acetyl groups from NH2-lysine of histones. This causes the electrostatic
attraction of positive histones with negative DNA.
DNA methyltransferases (DNMT) - enzymes catalyse the reaction of methyl group transfer from S-
adenosyl L-methionine (SAM) to a cytosine base in CpG dinucleotide.

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In eukaryotic cells, DNA methylation is responsible for tissue-specific regulation of gene
expression. Histone modifications (close and open their positive charge) and change
their binding with negative charge phosphodiester bond of DNA heterochromatin is
reversibly changed to euchromatin.

N-terminal tails of spinal histones usually contain a positive charge

K and R.

⚫ Modify N-terminal tails of core histones-K and R-close, open their positive
charge, and change their binding with the negative charge phosphodiester bond of
DNA.
⚫ Heterochromatin is reversibly changed to Euchromatin.
⚫ Histone acetylation is associated with higher transcriptional activity.

The reversible transformation from heterochromatin to euchromatin

results from histone modifications which affect their binding to DNA.
- Acetylation of histones H3 and H4
- Methylation of lysine (K) and arginine (R) residues at the N-terminus of histones H3
and H4
- Phosphorylation of serine (S) residues at the N-terminus of histones H2A, H2B, H3,
and H4
- Ubiquitination (u) of lysine residues at the C-terminus of histones H2A, and H2B

Epigenetic Regulation of Gene Expression

Epigenetics = inheritance of properties (e.g., gene expression) through mitosis, not
requiring a change to DNA sequence
Epigenetic mechanisms are associated
with chromatin remodelling and include: Epigenetic modifications play an essential role in the
- histone modifications regulation of gene transcription and are involved in
- DNA methylation reversibly rebuilding heterochromatin into euchromatin.
- nucleosome remodelling
- miRNA system in the cytosol

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DNA Transcription
Alpha-amanitin, a cyclic octapeptide, is toxic because of its affinity for RNA polymerase II in eukaryotic cells.
Since this enzyme is responsible for mRNA synthesis in the cell, the compound is a potent and selective
inhibitor of mRNA synthesis. In eukaryotic cells, we distinguish genes of type I, II, III, which DNA-dependent
RNA Polymerase transcribes
I, II, or III. RNA polymerases differ in their sensitivity to a-amanitin.

After converting heterochromatin into euchromatin in eukaryotic cells,

transcription factors-proteins begin to bind to the promoter.
A transcription factor (TF) = is a protein that controls the transcription rate of genetic
information from DNA to RNA by binding to a specific sequence -promoter. TFs bind to the DNA and help
initiate increased or decreased gene transcription programs. As such, they are vital for many critical cellular
processes.

Transcription factors form the essential transcription complex that allows for the
positioning of the RNA polymerase.

Homeobox genes encode some transcription factors.

▪ A homeobox = is a DNA sequence, around 180 base pairs long, found within genes involved in
morphogenesis in eukaryotes.
▪ Homeobox genes encode homeodomain protein products that are transcription factors sharing a
characteristic protein fold structure that binds DNA to regulate the expression of target genes.
▪ Homeodomain proteins regulate gene expression and cell differentiation during early embryonic
development.

Examples: the basic transcription complex, the activators that

interact with enhancers, the repressor that interacts with silencers
and coactivators.

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Protooncogene = a gene that is capable of triggering the process of neoplastic transformation.
- Proto-oncogenes are highly regulated genes that encode proteins mediating positive cell
growth regulation and cell survival signals.
- Mutations (changes) in a proto-oncogene may cause it to become an oncogene, which
can cause the growth of cancer cells.
- Oncogenes play a crucial role in carcinogenesis; their precursors are protooncogenes, e.g.,
receptors for polypeptide growth factors, growth factors, kinases involved in intracellular
signal transduction, and transcription factors.
- But after increasing their function, protooncogenes become oncogenes of continuous
activity, forcing malignant transformation.

Class I genes - include rRNA, located in the nucleolus

Class III - the eukaryotic promoter for the gene, is located in the cell
nucleus. Gene class III includes tRNA, 5S rRNA, snRNA, and snoRNA, transcribed by Pol III

The general structure of promoters for RNA polymerase II transcribes class II genes (pre-mRNA,
snRNA, snoRNA, miRNA) in eukaryotic cells.

Usually, eukaryotic promoters can be distinguished as the minimal promoter

(located several tens of base pairs from the transcription start site), the proximal, the promoter,
and the distal promoter.
In addition, transcription can be influenced by elements located outside the promoter, far away
from the gene, and several thousand base pairs from the start of transcription
(enhancers and silencers). They may be located upstream or downstream of the coding

Formation of the basic transcription complex, transcription of class II

genes encode pre-mRNA
- The essential transcription complex for RNA polymerase II (pol II) positioning includes
sequential assembly of the pre-initiation complex begins when the TFIID protein (which
contains TATA-box binding protein and TBP-associated factors) binds to a TATA box
sequence located -25 nucleotides from the start site transcription.
- TFIID also recognises and binds promoter sequences that do not contain the TATA
sequence.
- Then, it is joined by TFIIA and TFIIB. The next step is the binding of Pol II and TFIIF, TFIIE,
and finally TFIIH.
- TFIIH has helicase activity necessary for DNA strand separation in the promoter area and
elongation initiation.
- An associated kinase (TFIIK) can phosphorylate the CTD domain at the C-terminus of
RNA polymerase II.
- The CTD contains the Tyr-Ser-Pro-Thr-Ser-Pro-Ser repeats highly evolutionarily conserved
between different species.
- Phosphorylation of the CTD domain of RNA polymerase II activates polymerase II, which
starts to make primary transcript (pre-mRNA).

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Processing of pre-mRNA to mRNA
→ Addition, cap "at the end of 5.'
→ Polyadenylation at the end of 3.'
→ Splicing
→ Transport to the cytoplasm
→ Edition
→ Translation
→ Degradation

Post-transcription modifications of pre-mRNA in the cell nucleus

1. Attaching a guanylate cap at the 5 'end of the pre-mRNA. The guanylate cap is an unusual
nucleotide (7-methylguanosine). Finds the "protein factory" in the cytosol, facilitates binding to a small
ribosome subunit.
2. Attachment of the poly-A sequence at the 3 'end of pre-mRNA. The poly-A sequence is a short strand
of several dozen nucleotides with adenine. This mRNA is recognised as its own, not foreign, a nucleic
acid.
3. Splicing, i.e., removing introns and joining exons.
4. Editing of mRNA takes place in the cytosol and involves codon conversion, e.g., an amino acid in a stop
codon or an amino acid into another amino acid.

Mechanism of Splicing
- Alternative splicing = is the process of selecting different combinations of splice sites within a messenger
RNA precursor (pre-mRNA) to produce variably spliced mRNAs. These multiple mRNAs can encode
proteins that vary in their sequence and activity and yet arise from a single gene. Results in protein
isoforms.

Where is the mRNA inside eukaryotic cells degraded?

Processing bodies (P-bodies) enzymes are involved in mRNA turnover in the cytoplasm of the eukaryotic cell.
- Processing bodies (P-bodies) are distinct foci formed by phase separation
within the cytoplasm of the eukaryotic cell consisting of many enzymes
involved in mRNA turnover.
- P-bodies are highly conserved structures observed in somatic cells originating
from vertebrates, invertebrates, plants, and yeast.
- P-bodies have been demonstrated to play fundamental roles in general mRNA
decay, nonsense-mediated mRNA decay, adenylate-uridylate-rich element
mediated mRNA decay and microRNA-induced mRNA silencing

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DNA Transcription
By what motives do transcription factors bind to DNA -
chromatin?
Several motifs mediate the binding of regulatory proteins transcription factors to
DNA.

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