Genetics – chapter 9

Genetic code
Translation
Control of gene expression
GENETIC CODES
 One gene – one protein hypothesis
• Beadle and Tatum experiment on Neurospora to study the
biochemical result of mutations.
▫ The fungus can synthesize all the necessary metabolites
from the basic compounds (inorganic salts, nitrogen, a
carbon source such as sucrose, and the vitamin biotin)
▫ The fungus is mutated by radiation  the mutation is
cultured in different media supplied with different aa
▫ Mutant is compared with that cultured in complete medium
• The mutation of genes in the biosynthesis pathway
of arginine cause mutation in enzyme of the
metabolic pathway  fungal arginine deficiency.
•  one gene – one enzyme hypothesis
•  one gene – one polypeptide hypothesis
• The gene has information to define the polypeptide
and its functions  protein coding genes
Genetic code
• Regarding the number of nucleotide and number
of amino acid, the gene’s length is always longer
than the polypeptide’s length
• Two questions for the genetics codes:
▫ How many nucleotide is (are) corresponding for
one amino acid? 1? 2? 3? … or what?
▫ Which nucleotide for which amino acid?
How many Nu for synthesis of 1 aa
 there are about 20 aa in protein synthesis and
there are only 4 Nu in RNA
If each Nu codes for 1 aa  not enough to have
20 aa
If 2 Nu code for 1 aa  number of aa possibly
synthesized is 4 x4 = 16 aa  still not enough
If 3 Nu code for 1 aa  number of aa is 4x4x4 =
64 aa  this might be possible, because there
may be some sets of Nu code for the same aa
So the number of Nu in one codon is 3
Which Nu for which aa
 Several cell-free synthesis techniques were used
to find which Nu involved in synthesis of certain
aa
 Nirenberg and Matthaei used homopolymer and
random copolymer method
Nirenberg and Leder used ribosome bound tRNA
 see the correlation of codon in short RNAs and
aa in tRNA
Khorana used chemically synthesized repeated
RNA Nu in aa synthesis
 The genetic code was fully understood in 1968
Degeneracy of the code
• A codon consist of 3 Nu at either position (1st, 2nd, 3rd)
• In 64 codons:
▫ 3 used as stop codon, only 61 codons are sense codons
which code for amino acids
▫ Trp and Met are coded by single codon
▫ Some amino acids specified by 2 codons, some by more
(Gly 4 codons, Leu 6 codons)
• Isoaccepting tRNA: a aa is carried by different tRNAs
• Wobble: some codons differ at 3rd position and are
recognised by the same tRNA  synthesize the same
aa
Wobble
Reading frame and initiation codons
• Reading codon is non-overlapping
• For any sequence of Nu, there are 3 potential sets
of codon (3 ways the sequence is read to produce
polypeptide)  3 reading frames
• The reading frame is set by the initiation codon
(the first codon that synthesizes aa)
▫ initiation codon of a gene is usually AUG, sometimes
GUG or UUG are used
Termination codon
• 3 codons UAA, UAG and UGA do not code for aa
• no tRNA have anticodon that pair with these
codon
•  these codons signal the end of aa synthesis
The universality of the code

• Genetic code is
almost universal
that each codon
specify the same aa
in all organisms
• Some exceptions
are from the stop
codons
TRANSLATION
Translation occurs
through 3 stages
• Assembly at the
ribosome
• Enlongation
• Termination
Binding of aa to the tRNA

• Each tRNA is specifically

bound to 1 aa
• All tRNA has CCA at
the 3’ end  attach the
carbonyl group of aa to
the 2’ or 3’ OH of
adenine nucleotide
• There are about 30-50
tRNAs that carry about
20 aa
• Each aa is attached to
tRNA by its specific
enzyme aminoacyl –
tRNA synthetase
• Structure of ribosome
The initiation complex consists of
(1) The small subunit of the ribosome;
(2) The mRNA;
(3) The initiator tRNA with its amino acid (fMet-
tRNAfMet);
(4) One molecule of GTP; and
(5) IF-3, IF-2, and IF-1.
These components are collectively known as the 30S
initiation complex (c)
When the large subunit has joined the initiation
complex, it is called the 70S initiation complex
(d)
Initiation of translation
Initiation in prokaryote and eukaryote
Prokaryote Eukaryote
• The shine-Dalgano sequence • No Shine-Dalgano sequence,
of mRNA complements with instead, the small subunit of
16s rRNA in small subunit  ribosome recognize the cap of
locate mRNA to small subunit mRNA and bind to it  then
right at the start codon small subunit scan the mRNA
strand until it locate the start
codon which is nested in
Kozak sequence
• Require less initiation factors • Require more initiation factors
• Tail-binding protein interact
with cap-binding protein
enhance the binding of the
small subunit to 5’ end of
mRNA
• Binding of mRNA to subunit of ribosome in
prokaryote and eukaryote
Elongation
Enlongation requires:
(1) the 70S complex ;
(2) tRNAs charged with their amino acids;
(3) several elongation factors (EF-Ts, EF-Tu, and EF-G);
and
(4) (4) GTP.
• Enlongation occur in 3 steps:
▫ Bring charged tRNA to A site
▫ Create the peptide bond
▫ Translocation
Termination
When a stop codon is
encountered, release factors
associate with the ribosome
and bring about the
termination of translation.
There is no tRNA with
anticodon complement with
the termination codon
Release factors bind to the
stop codon and release
mRNA from ribosome
3. Post translational modification
• The formyl group or the entire methionine residue may be
removed from the amino end of a protein
• Some proteins are synthesized as larger precursor proteins and
must be cleaved and trimmed by enzymes before the proteins
can become functional.
• the attachment of carbohydrates may be required for activation
proper folding of the polypeptide chain;
• In eukaryotic cells, the amino end of a protein is often
acetylated after translation.
• removal of 15 to 30 amino acids, called the signal sequence
• Amino acids within a protein may also be modified:
• phosphates, carboxyl groups, and methyl groups are
• added to some amino acids.
Translation and antibiotics
REGULATION OF GENE EXPRESSION
 General principles of gene regulation
• Genes can be regulated at any level, including transcription, RNA
processing, translation, and post-translation.
• Control of transcription is an important mechanism of gene regulation.
• Transcriptional control can be negative (''on unless turned off") or positive
("off unless turned on"); many genes include regulatory regions for both
types of regulation.
• Most genes have multiple, overlapping regulatory mechanisms that
operate at more than one level, from transcription through post-
translation.
• In prokaryotes, the genes coding for the enzymes in a metabolic pathway
are often clustered in the genome and controlled jointly by a regulatory
protein that binds with an "operator" region at the 5' end of the cluster.
This type of gene organization is known as an operon.
• In eukaryotes, genes are not organized into operons. Genes at dispersed
locations in the genome are coordinately controlled by one or more
"enhancer" DNA sequences located near each gene that interact with
transcriptional activator proteins that enable transcription of each nearby
gene to occur.
• Level of gene control
▫ Alteration of gene structure (DNA methylation, chromatine
change …)
▫ Level of transcription for the cellular economy
▫ mRNA procesing (modified mRNA can be translated)
▫ RNA stability
▫ Level of translation
▫ Protein processing
• Gene and regulatory elements
▫ Structural genes: produce proteins for the metabolism
▫ Regulatory genes: produce RNA or proteins that interact
with other sequence (regulatory sequence) that affect the
transcription and translation
• DNA-binding proteins
▫ Bind to DNA and influence the expression of DNA
1. Gene regulation in bacterial cell
• Operon structure
▫ Genes that have related function are grouped and
are under control of 1 promoter
▫ Gene are often transcribed into one mRNA
▫ Regulatory gene produces regulatory protein that
binds to regulatory element and control the
expression of the structural gene a,b,c
• Negative and positive control: inducible and
repressible operon
▫ negative control: a regulatory protein acts as a
repressor, binding to DNA and inhibiting
transcription;
▫ positive control, a regulatory protein acts as an
activator, stimulating transcription.
Repressible regulation in Trp operon
• Trp operon is also controlled by attenuation
▫ Repression is never complete; some transcription
is initiated even when the trp repressor is active;
repression reduces transcription only as much as
70-fold.
▫ Attenuation can further reduce transcription
another 8- to 10-fold; so together the two
processes are capable of reducing transcription of
the trp operon more than 600-fold.
• Antisense RNA in gene regulation
▫ Beside protein regulators, many RNA regulators
have been discovered
▫ Small RNA molecules bind by complement to the
particular sequence on mRNA and control the
gene expression  antisense RNA
▫ Example: antisense RNA control the expression of
ompF (produce the outer membrane protein that
functions as membrane pore that allow bacterium
to adapt the environment osmolarity)
2. Eukaryotic gene regulation
• Some common features seen in both prokaryote and
eukaryote: controlled by DNA binding proteins, contains
regulatory elements…
• Different features:
▫ Gene is not structured in operon, instead, each gene has its own
promoter
▫ Chromatin structure affects gene expression
▫ Activators are more common in eukaryotic system than in
prokaryote
▫ Regulation of gene expression in eukaryotic cells is
characterized by a greater diversity of mechanisms that act at
different points in the transfer of information from DNA to
protein.
Chromatin structure and gene regulation
• For a gene to be transcribed, DNA segment should be released
from histone complex.
▫ This relaxation allow DNAseI hydrolyse gene  DNAseI
hypersensitivity region (this is also region for biding with
regulatory factor)
• Histone acetylation
▫ Acetyl groups are added to the histone proteins by acetyl
transferase  destablize nucleosome structure  allows DNA
separate from histone
▫ Enzyme deacetylase strip acetyl group from histone  bring back
the structure of nucleosome
• DNA methylation
▫ Heavily methylated DNA is associated with the
repression of transcription in vertebrates and plants,
whereas transcriptionally active DNA is usually
unmethylated in these organisms.
▫ DNA methylation is most common on cytosine bases
adjacent to guanine nucleotides on the same strand
(CpG);
▫ Association between DNA methylation and the
deacetylation of histones, both of which repress
transcription
Transcriptional Control in Eukaryotic Cells
• Transcriptional activators,
coactivators and repressors
• Enhancers and insulators
• Coordinated gene regulation
▫ several eukaryotic genes
may be activated by the
same stimulus
Gene Control Through Messenger
RNA Processing

• Alternative splicing
allows a pre-mRNA
to be spliced in
• multiple ways,
generating different
proteins in different
tissues
• or at different times
in development
Gene Control Through RNA Stability
• there is great variability in the stability of
eukaryotic mRNA: some persist for only a few
minutes; others last for hours, days, or even
months  result in large differences in the
amount of protein that is synthesized.
RNA Silencing
• expression of some genes
may be suppressed
through RNA silencing,
also known as RNA
interference and
posttranscriptional gene
silencing
Translational and Posttranslational Control
• The initiation of translation in some mRNAs is regulated
by proteins that bind to the mRNA’s 5 UTR and inhibit
the binding of ribosomes, similar to the way in which
repressor proteins bind to operators and prevent the
transcription of structural genes.
• Many eukaryotic proteins are extensively modified after
translation by the selective cleavage and trimming of
amino acids from the ends, by acetylation, or by the
addition of phosphates, carboxyl groups, methyl groups,
and carbohydrates to the protein)  affect the transport,
function, and activity of the proteins and have the
capacity to affect gene expression.