GENOME

GENE EXPRESSION
 GENOME
- term was formed from gene and chromosome in 1920 by Hans Winkler
total complement of genetic material contained in an
organism or cell (DNA or RNA)
includes both genes and non-coding sequences of DNA
complete DNA sequence of one set of chromosomes

NUCLEAR genome (complete set of nuclear DNA)

MITOCHONDRIAL genome
CHLOROPLAST genome
PLASMID genome

GENOMICS - study of genomes of related organisms

Many genomes were sequenced by genome projects:

first DNA-genome project was completed in 1977
Many genomes were sequenced by genome projects:
first DNA-genome project was completed in 1977

Human Genome Project

was organized to map and to sequence human genome (2001)

Other genome projects

include mouse, rice, plant Arabidopsis thaliana,
puffer fish, bacteria E. coli, etc.

Organism Genome size (base pair) Gene number

Bacteriophage X174 5386 10
Escherichia coli 4106 2 350
Drosophila melanogaster 1.3108 13 601
Homo sapiens 3.2109 39 114

Genome size and number of genes vary from one species to another !!!
 GENE
MENDEL (1865) - used term discreet elements"
JOHANSSEN (1909) - first used the term gene
MORGAN (1911) - gene is located on locus of chromosome
BEAGLE, TATUM (1941) - one gene encodes one enzyme
WATSON, CRICK (1953) - gene is part of DNA

GENE
unit of heredity in living
organisms, encoded in a
sequence of nucleotide that
make up a strand of DNA
gene can have multiple different
forms = alleles (defined by
different sequences of DNA)
Structure of a gene
genes have a 5 to 3 orientation
top strand is coding strand, bottom strand is called template
strand (complementary)
gene is composed of:
1) PROMOTER (with TATA box) - recognized by the
transcription machinery
2) regions that code for a protein or RNA
3) ENHANCERS = regulatory sequence for regulation of
promoter (upstream or downstream, sometimes even within an intron of
the transcribed gene)

GENE
proximal proximal distal

promoter
TATA box
highly conserved sequence, usually followed by three or more
adenine bases: 5'-TATAAA-3'
in promoter region of most genes in eukaryotes and archea
binding site for transcription factors or histones
(the binding of a transcription factor blocks the binding of a histone and reverse)
binding site for RNA polymerase

upstream
 How gene can look like?

Animation of ORF:
http://www.learner.org/courses/biology/archive/
OPEN READING FRAME (ORF) animations/hires/a_genom6_h.html

sequence of bases that could potentially encode a protein

located between the start-code sequence (initiation codon) and
the stop-code sequence (termination codon)
EUKARYOTIC GENE
composed of exons (coding sequence of gene) and introns
(non-coding sequence of gene, much longer, transcribed
but never translated into protein)
one gene is transcribed to one mRNA !!

CCAAT box (CAAT box or CAT box) for binding of transcription factors
(in eukaryotic cells)
PROKARYOTIC GENE
simple gene without introns !!!
organized into operons (groups of genes), whose products
have related functions and are transcribed as a unit
several genes are transcribed to one mRNA
 GENE EXPRESSION
DNA RNA PROTEIN (phenotypic trait)

DNA
TRANSCRIPTION
post-transcription
modification
mRNA

TRANSLATION
post-translation
protein
modification

phenotype
(trait)
 TRANSCRIPTION
Three phases: 1) initiation, 2) elongation, 3) termination

1. INITIATION
transcription factors (50 different proteins) bind to promotor
sites on the 5 side of gene to be transcribed
RNA polymerase (enzyme) binds to the complex of
transcription factors by help of sigma factor (subunit of RNA
polymerase)
working together, they open the DNA double helix
RNA polymerase begins the synthesis of RNA at a specific
sequence initiation site (start signal)
ribonucleotides are inserted into the growing RNA strand
following the rules of base pairing
removing of sigma factor
 2. ELONGATION
synthesis of the RNA proceeds in the 5 3 direction
 3. TERMINATION
recognition of termination sequence (terminator = 4 - 8
adenines) in DNA
when the polymerase moves into the region of terminator,
hairpin loop is formed in mRNA
hairpin loop pulls away from DNA (weak bonding between
adenine run in DNA and uracils in mRNA)
transcript is released from RNA polymerase and RNA
polymerase is released from DNA

In prokaryotes signal
for RNA polymerase to
release is binding to rho
protein
Animation of transcription:
*http://highered.mcgraw-
hill.com/sites/0072835125/student_view0/anim
ations.html#
Stages of transcription
*http://www-
class.unl.edu/biochem/gp2/m_biology/animatio
n/gene/gene_a2.html

http://bcs.whfreeman.com/thelifewire/content/c
hp12/1202001.html
 Prokaryotic vs. eukaryotic transcription
Eukaryotic transcription
- localized to the nucleus, while translation occurs in cytoplasm
- mRNA carry information of one gene

Prokaryotic transcription
- occurs in the cytoplasm alongside translation
- polycistronic mRNA carry information of more genes (operon)
 POST-TRANSCRIPTION MODIFICATION
PROCESSING of pre-mRNA in Eukaryotes:
- primary transcripts produced in nucleus must undergo steps to
produce functional RNA molecules for export to the cytosol
Animation of splicing:

pre-mRNA mRNA
*http://bcs.whfreeman.com/thelifewire/con
tent/chp14/1402001.html

http://highered.mcgraw-
hill.com/sites/0072835125/student_view0/

The steps of RNA processing: animations.html#

1. synthesis of cap (modified guanine attached to 5end of pre-mRNA)

protects RNA from being degraded by enzymes
serves as an assembly point for proteins that recruit the small
subunit of ribosome to begin translation
2. removal of introns and splicing of exons by spliceosome
= complex of snRNP molecules ("snurps", small nuclear
ribonucleoproteins) and 145 different proteins
(introns begin with GU and end with AG)
2. synthesis of poly(A) tail (100-200 adenine nucleotides attached to
3end of pre-mRNA )
Cutting and splicing of mRNA must be done with precision
- if even one nucleotide is left over from intron or one is removed from exon,
the open reading frame will be shifted, producing new codons and different
sequence of amino acids
 POST-TRANSCRIPTION MODIFICATION

Processing of pre-rRNA pre-rRNA rRNA

pre-rRNA is a cluster of 3 rRNAs (18S, 5.8S, 28S) synthetized
in nucleolus, separated by snRNA (small nuclear RNA) to
become functional
5S rRNA is synthesized in nucleoplasm, then enters
nucleolus to combine with 28S and 5.8S rRNAs, forming
large subunit of ribosome

Ribosome in prokaryotes (70S)

small subunit (30S) - 16S +
proteins
large subunit (50S) - 5S, 23S
+ proteins
Ribosome in eukaryotes (80S)
small (40S) - 18S + proteins
large (60S) - 5S, 28S, 5.8S +
proteins
 POST-TRANSCRIPTION MODIFICATION
Processing of pre-tRNA
pre-tRNA is synthesized in
nucleolus
1) cleavage - removing of extra
segment at 5' end
2) splicing - removing intron in
anticodon loop
3) addition of CCA at 3'end
(found in all mature tRNAs)
4) base modification - some
residues are modified to
characteristic bases

pre-tRNA tRNA
 GENETIC CODE
1966 - Nirenberg, Khoran, Ochoa (1968 Nobel price)

each cell within our bodies contains genetic blueprint

(genome composed of DNA)
DNA is linear sequence of four-letter alphabet with A,C,G,T
(adenine, cytosine, guanine, thymine)
inside of a gene-coding region, every 3 bases (codons)
codes for amino acid, there are 4 possibilities for each
position, 4*4*4 = 64 potential three-base patterns

AUG the begin of translation (encodes methionin)

UAA, UAG, UGA (stop codons) the end of translation
60 codons - for 20 amino acids (redundancy in genetic code)
 Redundancy in genetic code
In DNA

In RNA
 RIBOSOME
composed from rRNA and proteins (in ratio 1:1)
found in cytoplasm of prokaryotic and eukaryotic cells
found in matrix of mitochondria and in stroma of chloroplasts
can be free or fixed to membranes of ER
104-105 ribosomes in cell

Svedberg coefficient (S)

- non-SI physical unit
- after Theodor Svedberg, Nobel prize
- for measurement of relative size of
particle by rate of sedimentation in
centrifugal field (bigger particles have
higher values); 1 svedberg = 1013s
- when 2 particles bind together there is
loss of surface area (70S ribosome is
composed of 50S subunit and 30S subunit)
 Prokaryotic Eukaryotic
Ribosome 70 S 80 S
Large subunit 50 S 60 S
Small subunit 30 S 40 S
Ribosome with four specific binding sites:
mRNA binding site
A (aminoacyl-tRNA binding site)
P (peptidyl-tRNA binding site)
E (exit site)

Espero Publishing, s.r.o.

polysom - a cluster of ribosomes connected by a strand of
mRNA and actively synthetizing protein
(in prokaryotic and eukaryotic cells)

stop codon

start codon

mRNA

polypeptide

*Animation of
polysome:
http://www.suma
nasinc.com/webc
ontent/anisample
s/molecularbiolo
gy/polyribosome
s.html
 tRNA
structure is similar to a clover leaf
anticodon (triplet of bases complementar to codon on mRNA)
enzyme amino acyl tRNA synthetase recognizes specific
tRNAs and catalyzes the attachment of the appropriate
amino acid to the 3end (20 synthetases for 20 aminoacids)
 TRANSLATION

1. INITIATION
initiator tRNA with initiation factors
binds to small ribosomal subunit
initiator tRNA moves along mRNA
searching for first start codon AUG
initiator factors dissociate
large ribosomal subunit binds

energy from GTP

Types of initiator tRNA:

- METHIONINE (in archea, eukaryotes)
- FORMYLMETHIONINE (eubakteria)
 2. ELONGATION
second tRNA enters the ribosome (A site)
and attaches to its complementary
mRNA codon
tRNA moves from A to P and to E sites
aminoacids binds together by peptide bond,
that is formed by a ribozyme (enzyme
composed of RNA)
ribosome moves along mRNA and protein
grows longer
elongation factors
energy from GTP
 3. TERMINATION
translation ends with stop codons UGA, UAA, UAG
no tRNA exists that recognizes stop codon in mRNA, instead
release factor (termination factor) enters the ribsome
peptide chain is released from tRNA and leaves the ribosome
ribosome dissociates into its large and small subunits
protein synthesis is completed

Animation of translation:
*http://www.biostudio.com/demo_freeman_protein_synthesis.htm
-http://www.phschool.com/science/biology_place/biocoach/translation/addaa.html
- http://bcs.whfreeman.com/thelifewire/content/chp12/1202003.html
- http://highered.mcgraw-hill.com/sites/0072835125/student_view0/animations.html#
 POST(CO)-TRANSLATION MODIFICATION
chemical modification of primary structure
proteolytic cleavage - removing of amino acids
phosphorylation for controlling the behavior of a protein
attaching functional groups (glycosylation, sulfation.)
formation of disulfide bridges

formation of secondary, tertiary,

quaternary structures
spontaneously
by chaperons (Hsp heat shock proteins)
 REGULATION OF GENE EXPRESSION
in prokaryotes Animation of regulation:
http://highered.mcgraw-
hill.com/sites/0072835125/student
_view0/animations.html#
1. Constitutive expression
in genes encoding proteins required for life under all conditions
amount of constitutive protein produced depends on promoter
no additional factors are required
2. Adaptive expression
regulation of transcription by
repressor
 REGULATION OF GENE EXPRESSION
in eukaryotes

5 6 7
1 2 3 4

1. Modification of DNA and histone

DNA methylation - gene silencing
histone acetylation - gene silencing
2. Transcriptional control
each eukaryotic gene needs its own promoter (DNA sequence
responsible for initiating low levels of transcription and determining
the transcription start site)
eukaryotic genes are primarily regulated by transcriptional
activators (transcription factors)
eukaryotic genes have one or more enhancers (DNA sequences
associated with the gene being regulated, responsible for increasing
"enhancing" transcription levels, and for regulating cell- or tissue-
specific transcription) Animation of transcription:
http://bcs.whfreeman.com/thelifewire/conten
t/chp14/1402002.html
3. RNA interference (RNAi)
- historically post-transcriptional gene silencing
- conserved in most eukaryotic organisms
2006 - Andrew Fire and Craig C. Mello (Nobel prize)

RNAi pathway is initiated by enzyme dicer, which cleaves

dsRNA to short ds fragments of siRNA (small interfering
RNA of 2025 base pairs)
siRNA is incorporated into RNA-induced silencing complex
(RISC)
RISC binds to mRNA and induces its degradation by
enzyme slicer (catalytic component of RISC)

ds DNA mRNA + RISC (siRNA + enzyme slicer)

degradation of mRNA blocking of translation
 RNA interference

RNAi can be used for large-scale

screens that systematically shut down
genes in the cell to identify the
components necessary for a particular
cellular process

promising tool in biotechnology and

medicine

Animation of interference:
http://highered.mcgraw-
hill.com/sites/0072835125/student_view0/
animations.html#
4. RNA processing control
splicing
5. RNA transport control
6. Regulation of translation
initiation, elongation and
termination factors
7. Protein activity control
modulation of chemical
modification of proteins
modulation of proteolysis
(ubiquitination)
 UBIQUITINATION
regulated degradation of proteins in the cell, ATP is needed

UBIQUITIN conserved small protein (76 amino acids), used to

target proteins for destruction
PROTEASOME complex of enzymes that degrades
endogenous proteins (transcription factors, cyclins, proteins
encoded by viruses and intracellular parasites, proteins that are not
correctly translated)
SUMO proteins compete for binding sites with ubiquitin,
influence protein activity, do not lead to their degradation

Proteasome - in all organisms

Ubiquitination - only in eukaryotes !!!
 UBIQUITIN is activated by enzyme E1
ACTIVATED UBIQUITIN is added by enzyme E2 to the
substrate (target protein)
other 3 ubiquitins are added by enzyme E3 (ubiquitin-
ligase)
protein marked by ubiquitins is degradated in PROTEASOME
 ONTOGENESIS (DEVELOPMENT) AND GENES
HOMEOBOX GENES
involved in the regulation of development (morphogenesis) in
animals, fungi and plants
encode transcription factors (proteins) that bind to DNA at
specific promoter or enhancer and thus regulate
transcription

HOMEOBOX
DNA sequence (180 bp long) found within homeobox genes
it encodes a protein domain (homeodomain) belonging to
a transcription factors and acts as an "on/off" switch
for gene transcription
HOX GENES
first identified in Drosophila melanogaster
highly conserved subgroup of
homeobox genes
determine the longitudinal axis
of the body plan
as regulatory genes they
establish the identity of
particular body regions

Hox genes determine the location

of body segments in a developing
fetus or larva
 MUTATIONS
Mutations in homeobox genes alter gene regulation, and hence
cause phenotypic changes important in evolution !!!

In normal flies: structures like legs, wings, and antennae

develop on particular segments, and this process requires
the action of homeotic genes
In mutant flies: structures characteristic of one part of embryo
are found at some other location as Antennapedia
Normal head Fly with the dominant
Antennapedia mutation
- legs where the antennae
should be!

Animation of hox genes:

http://bcs.whfreeman.com/thelifewire/c
ontent/chp21/21020.html