The Central Dogma
All organisms, from the simplest bacteria to human beings, use the same basic mechanism of reading and expressing genes. This is so fundamental to life and it is known as the “Central Dogma”. Information passes from the genes (DNA) to an RNA copy of the gene, and the RNA copy directs the sequential assembly of a chain of amino acids or protein.
DNA
® RNA ® Protein
The information encoded in genes is
expressed to protein in two phases: transcription,
in which RNA polymerase assembles an mRNA molecule complementary to the DNA and
translation, in which a ribosome
assembles a polypeptide, whose amino acid sequence is specified by the
nucleotide sequence in mRNA.
Transcription: An
Overview
The
first step of the Central Dogma is the transfer of information from DNA to RNA,
which occurs when an mRNA copy of the gene is produced. This stage is called transcription.
Transcription is initiated when the enzyme RNA polymerase binds to a
particular binding site called a promoter located at the beginning of a
gene. Starting there, the RNA polymerase moves along the strand into the gene.
As it encounters each DNA nucleotide, it adds the corresponding complementary
RNA nucleotide to a growing mRNA strand. Thus, guanine (G), cytosine (C),
thymine (T), and adenine (A) in the DNA would signal the addition of C, G, A,
and uracil (U), respectively, to the mRNA.
When
the RNA polymerase arrives at a transcriptional “stop” signal at the end of the
gene, it disengages from the DNA and releases the newly assembled RNA chain.
This chain is the mRNA which is the complementary transcript of the gene from
which it was copied.
Translation:
An Overview
The
second step of the Central Dogma is the transfer of information from RNA to
protein. Here the information contained
in the mRNA is used to direct the sequence of amino acids during the synthesis
of polypeptides by ribosomes. Translation begins when an rRNA molecule within
the ribosome recognizes and binds to a “start” sequence on the mRNA. The
ribosome then moves along the mRNA molecule, three nucleotides at a time. Each
group of three nucleotides is a code word that specifies which amino acid will
be added to the growing polypeptide chain. The ribosome continues in this
fashion until it encounters a translational “stop” signal; then it disengages
from the mRNA and releases the completed polypeptide.
Kinds
of RNA
The
class of RNA found in ribosomes is called ribosomal RNA (rRNA). During
polypeptide synthesis, rRNA provides the site where polypeptides are assembled.
Transfer
RNA (tRNA) molecules both transport the amino
acids to the ribosome for use in building the polypeptides and position each
amino acid at the correct place on the elongating polypeptide chain.
Messenger
RNA (mRNA) molecules are long strands of RNA
that are transcribed from DNA and that travel to the ribosomes to direct
precisely which amino acids are assembled into polypeptides.
t RNA |
The genetic code
The
genetic code is the key that relates, in Crick’s words, “...the two great
polymer languages, the nucleic acid language and the protein language.”
The
“letters” in the “language” were found to be the bases; the “words” (codons)
are groups of bases; and the “sentences” and “paragraphs” equate with groups of
codons.
The
basic problem of such a genetic code is to indicate how information written in
a four-letter language (four nucleotides or nitrogen bases of DNA) can be
translated into a twenty-letter-language (twenty amino acids of proteins).
The
group of nucleotides that specifies one amino acid is a code word or codon. The
simplest possible code is a singlet code (a code of single letter) in which one
nucleotide codes for one amino acid. Such a code is inadequate for only four
amino acids could be specified. A doublet code (a code of two letters) is also
inadequate because it could specify only sixteen (4 × 4) amino acids, whereas a
triplet code (a code of three letters) could specify sixty four (4 × 4 × 4)
amino acids and Four-base code (4 × 4 × 4× 4) specify 256 combinations.
Crick
and his colleagues reasoned that it will be a triplet code (a code of three
letters) since it is more than enough to code for the 20 amino acids. Their
confusion was whether the codons are continuous sequence of transcribed
nucleotides or the sequence is punctuated with untranscribed nucleotides
between the codons, like the spaces that separate the words in a sentence. Crick and his colleagues used a chemical to
delete one, two, or three nucleotides from a viral DNA molecule (T4 bacteriophages
of E. coli) and then analysed whether
a gene downstream of the deletions was transcribed correctly. When they made a
single deletion or two deletions near each other, the reading frame of the
genetic message shifted, and the downstream gene was transcribed as nonsense.
However, when they made three deletions, the correct reading frame was
restored, and the sequences downstream were transcribed correctly. They
obtained the same results when they made additions to the DNA consisting of
one, two, or three nucleotides.
Thus
it was concluded that the genetic code is read as three nucleotides (in other
words, it is a triplet code) and that reading occurs continuously without
punctuation between the three-nucleotide units.
In
1961, Marshall Nirenberg discovered that adding the synthetic mRNA molecule
polyU (an RNA molecule consisting of a string of uracil nucleotides) to
cell-free systems resulted in the production of the polypeptide
polyphenylalanine (a string of phenylalanine amino acids). Therefore, UUU
specify phenylalanine.
In
1964, Nirenberg and Philip Leder developed a triplet binding assay in which a specific triplet was tested to see
which radioactive amino acid (complexed to tRNA) it would bind. 47 of the 64
possible triplets gave unambiguous results.
Har
Gobind Khorana decoded the remaining 17 triplets by constructing artificial
mRNA molecules of defined sequence and examining what polypeptides they
directed. In these ways, all 64 possible three-nucleotide sequences were
tested, and the full genetic code was determined.
Some of the most
important properties of genetic codes are as follows:
1. The code is a triplet
codon:
The
nucleotides of mRNA are arranged as a linear sequence of codons, each codon
consisting of three successive nitrogenous bases, i.e., the code is a triplet
codon.
2. The code is
non-overlapping:
In
translating mRNA molecules the codons do not overlap but are “read”
sequentially. Thus, a non-overlapping code means that a base in mRNA is not
used for different codons. However, it
has been shown that in the bacteriophage ɸ × l74 there is a possibility of
overlapping codons.
3. The code is commaless:
The
genetic code is commaless, which means that no codon is reserved for
punctuations. It means that after one amino acid is coded, the second amino
acid will be automatically, coded by the next three letters and that no letters
are wasted as the punctuation marks.
4. The code is
non-ambiguous:
Non-ambiguous
code means that a particular codon will always code for the same amino acid. In
case of ambiguous code, the same codon could code two or more than two
different amino acids.
5. The code has polarity:
The
code is always read in a fixed direction, in 5′→3′ direction. In other words,
the codon has a polarity. If the code is read in opposite directions, it would
specify two different proteins.
6. The code is
degenerate:
More
than one codon may specify the same amino acid; this is called degeneracy of
the code. For example, except for tryptophan and methionine, which have a
single codon each, all other 18 amino acids have more than one codon.
Phenylalanine,
tyrosine, histidine, glutamine, asparagine, lysine, aspartic acid, glutamic
acid and cysteine have two codons each.
Isoleucine
has three codons.
Valine,
proline, threonine, alanine and glycine have four codons each.
Leucine,
arginine and serine have six codons each.
The
code degeneracy is of two types: partial and complete. Partial degeneracy
occurs when first two nucleotides are identical but the third nucleotide of the
degenerate codons differs, e.g., CUU and CUC code for leucine. Complete
degeneracy occurs when any of the four bases can take third position and still
code for the same amino acid (e.g., UCU, UCC, UCA and UCG code for serine).
7. Some codes act as
start codons:
In
most organisms, AUG codon is the start or initiation codon, i.e., the
polypeptide chain starts either with methionine (eukaryotes) or N-
formylmethionine (prokaryotes).
In
rare cases, GUG also serves as the initiation codon, e.g., bacterial protein
synthesis.
8. Some codes act as stop
codons:
Three
codons UAG, UAA and UGA are the chain stop or termination codons. They do not
code for any of the amino acids. These codons are not read by any tRNA
molecules (via their anticodons), but are read by some specific proteins,
called release factors (e.g., RF-1, RF-2, RF-3 in prokaryotes and RF in
eukaryotes). These codons are also called nonsense codons, since they do not
specify any amino acid.
The
UAG was the first termination codon to be discovered and was named amber. UAA
is termed ochre and UGA is opal or umber.
9. The code is universal:
Same
genetic code is found valid for all organisms ranging from bacteria to man. The
genetic code may have developed 3 billion (300 crore) years ago with the first
bacteria, and it has changed very little throughout the evolution of living
organisms.
Recently,
some differences have been discovered between the universal genetic code and
mitochondrial genetic code. In the case
of yeast mitochondria, UGA codes for tryptophan, while in the nuclear genes,
UGA is a termination codon.
Wobble hypothesis
According
to this hypothesis, the first two bases of the anticodon are strictly standard
for the first two constant bases of the codon and pair strongly with them. The third
base of the codon is not so specific in its base pairing and may wobble (pair
loosely) in pairing with the corresponding base in the anticodon. As a result
of this, each tRNA recognizes several codons for its amino acid.
If
U is present at first position of anticodon, it can pair with either A or G at
the third position of codon. Similarly, when C or A occurs in the 5′ position
of the anticodon, it can pair only with G or U, respectively, in the 3′
position of a codon.
A wobble base pair is a pairing between two nucleotides in RNA molecules that does not follow Watson-Crick base pair rules.
Transcription
The
first step in gene expression is the production of an RNA copy of the DNA
sequence encoding the gene, a process called transcription. The enzyme involved is RNA polymerase.
Only
one of the two strands of DNA, called the template strand, is transcribed. The
RNA transcript’s sequence is complementary to the template strand. The strand
of DNA that is not transcribed is called the coding strand. It has the same
sequence as the RNA transcript, except T takes the place of U. The coding
strand is also known as the sense (+) strand, and the template strand as the
antisense (–) strand. In both bacteria
and eukaryotes, the polymerase adds ribonucleotides to the growing 3′ end of an
RNA chain. No primer is needed, and synthesis proceeds in the 5′→3′ direction.
Prokaryotic Transcription
A
prokaryotic protein coding gene is divided into three sequences with respect to
transcription.
1. A
sequence adjacent to the start of the gene called the Promoter with which RNA
Polymerase (RNAP) interact.
2. The
RNA coding sequence. The sequence of DNA
base pairs transcribed by RNAP into single stranded mRNA transcript.
3. A
sequence adjacent to the end of the gene where transcription will stop. This sequence is known as terminator
sequence.
The
promoter is considered upstream from the gene and the terminator is downstream
from the gene.
Factors required for
transcription
Bacterial
RNA polymerase is very large and complex, consisting of five subunits: two α
subunits bind regulatory proteins, a β′ subunit binds the DNA template, a β
subunit binds RNA nucleoside subunits, and a σ subunit recognizes the promoter
and initiates synthesis.
1.
RNA polymerase (enzyme that catalyzes the synthesis of RNA from a DNA
template).
a) Core enzyme = 3 different types of subunits (2 α; 1
β; 1 β′)
(1)
β - binds incoming nucleotides
(2)
β′ – binds DNA
(3)
α - helps with enzyme assembly; interacts with other transcriptional activator
proteins
b) Holoenzyme = core enzyme + σ factor
σ factor recognizes the promoter
a) Can bind to specific DNA sequences and help RNA
polymerase initiate transcription via protein-protein interactions or by
altering the structure of the DNA.
b) Transcription of some promoters requires an
accessory transcriptional activator; at other promoters, the activators just
increase the rate of transcription but are not absolutely required.
3.
Template DNA containing gene or genes to be transcribed
4.
Promoter - The regulatory element that determine when a gene “turned on”
(transcribed) or “turned off”. The promoter DNA is located upstream of the gene
and contains a sequence which σ factor of RNAP and other transcription factors
bind. The -35 region and the -10 region
comprise the prokaryotic promoter.
·
The consensus sequence
(sequence indicating which nucleotides are found most frequently) for -35
region is 5’ TTGACA 3’.
·
The consensus sequence
for -10 region (Pribnow box) is 5’ TATAAT 3’.
The Promoter is recognized and bound by RNA polymerase during
initiation. This region of the DNA is the first place where base pairs separate
during transcription to allow access to the template strand. The AT-richness of
Pribnow box is important to allow this separation, since adenine and thymine
are easier to break apart.
5.
NTPs, Mg2+
Prokaryotic
Transcription stages
I.
Initiation
1.
RNAP (Holoenzyme) scans the DNA looking for promoters.
4. RNAP binds to double helical DNA covering
approximately 60 basepairs forming a closed complex.
5. RNAP unwinds the DNA resulting in open complex
formation.
6. First nucleotides are added to start RNA chain.
7. Accessory transcription factors may aid in the
steps.
II.
Elongation
1. Elongation is 5′→3′
2. σ factor is ejected from RNAP after first 2-10
nucleotides are added.
3. It was once believed that elongation occurred at a
constant rate; however, recent work suggests that RNAP may pause during
elongation.
III.
Termination (2 types)
1. Rho independent: A specific sequence at the end of
the gene signals termination. The sequence is transcribed into RNA. This
sequence contains numerous Gs and Cs, which forms a hairpin structure, followed
by a string of Us.
The hairpin destabilizes the DNA: RNA hybrid leading
to dissociation of the RNA from the DNA.
2.
Rho dependent: Rho protein binds to a sequence in the RNA known as rut site.
Rho moves along the RNA in the 3’ direction until it eventually unwinds the
DNA:RNA hybrid in the active site, thereby pulling the RNA away from the DNA
and RNAP.
Eukaryotic
transcription occurs in nucleus
In eukaryotes RNA polymerase II transcribes protein coding genes or structural genes. The product of transcription is precursor mRNA or Pre-mRNA. This molecule must be modified and/ or processed to produce the mature and functional mRNA molecule.
Factors involved
1.
RNA polymerases – Much more complex that prokaryotic RNAP (numerous additional
factors required, multiple polymerases)
|
Type
|
Location
|
RNA
synthesized |
Effect
of α-amanitin |
|
I |
Nucleolus |
5.8 S, 18 S,
and 28S rRNAs |
Insensitive |
|
II |
Nucleus |
mRNA, hnRNAs, some snRNAs |
Highly Sensitive |
|
III |
Nucleus |
tRNAs, 5S rRNA, some snRNAs |
Intermediate Sensitivity |
α-amanitin is a toxin produced by the mushroom Amanita phalloides.
2.
Eukaryotic RNAPs have subunits that are homologous to α, β, and β’ of
prokaryotic RNAP; however, eukaryotic RNAP also contain many additional
subunits.
3.
Template DNA containing the gene to be transcribed
4.
Eukaryotic regulatory elements
A
protein coding gene has several sequences involved in the regulation of gene’s
transcription. There will be positive regulatory elements (for activation
transcription) and negative regulatory elements (for repressing transcription).
Specific transcription factors (required for initiation of transcription) and
regulatory factors (involved in the activation or repression of transcription)
bind to these elements. The regulatory
elements will be several hundred bases away from the site of initiation o
transcription. The regulartory elements
adjacent to transcription start site are known as promoters and the distant
regulatory elements are known as enhancers.
a. Closest
to the transcription start site is the TATA box or TATA element or
Goldberg-Hogness box located –25 to -30 from the start of transcription. It has the consensus sequence 5’ TATAAA 3’.
b. Upstream
from the TATA region is a variably located sequence containing the sequence
CCAAT (frequently at –80) known as CAAT box.
It has the consensus sequence 5’ GGCCAATCT 3’.
c. Then
GC box has the consensus sequence 5’ GGGCGG 3’.
This helps RNA polymerase bind near the transcription start point.
4. NTPs,
Mg2+
Transcription events in
eukaryotes
RNAP
is unable to recognise the promoter region.
It needs Transcription Factors (TFs) to bring about the initiation of
transcription by RNAP. TF1 for RNAPI,
TFII for RNAPII and TFIII for RNAPIII.
During initiation TFIID or TATA factor
binds to the TATA element and TFIIA and a regulatory factor which is bound to
enhancer sequence. This binding makes
the DNA to form a loop and RNAP can bind and along with TFIIB, TFIIF and TFIIE
forms the transcription initiation complex.
During elongation, TFIIB and TFIIE are
released. TFIIS binds to the polymerase
enzyme and elongation continues.
The last stage of transcription is
termination, which leads to the dissociation of the complete transcript and the
release of RNA polymerase from the template DNA. The process differs for each of
the three RNA polymerases. The
termination of transcription of pre-rRNA genes by polymerase I is performed by
a system that needs a specific transcription termination factor and the
mechanism bears some resemblance to the rho-dependent termination in
prokaryotes. In case of polymerase II,
two protein complexes recognize the poly-A signal in the transcribed RNA. The long poly-A tail is unique to transcripts
made by Pol II. RNA polymerase III can
terminate transcription efficiently without involvement of additional factors.
The Pol III termination signal consists of a stretch of thymines (on the non template
strand) located within 40bp downstream from the 3' end of mature RNAs. The poly-T termination signal pauses Pol III
and causes it to back track to the nearest RNA hairpin to become a “dead-end”
complex
Post transcriptional
modifications or RNA processing
Post-transcriptional
modification or Co-transcriptional modification is a process in eukaryotic
cells by which, primary transcript RNA or Pre-mRNA is converted into mature
RNA. This process is vital for the correct translation of the genes in
eukaryotes because the human primary RNA transcript contains both exons (coding
sections of the transcript) and introns (non-coding sections of the primary RNA
transcript).
The
pre-mRNA molecule undergoes three main modifications. These modifications are
5' capping, 3' polyadenylation, and RNA splicing, which occur in the cell
nucleus before the RNA is translated.
1. 5’
capping: At the end of transcription, the 5' end of the RNA transcript contains
a free triphosphate group. The capping process replaces the triphosphate group
with another structure called the "cap". The cap is added by the
enzyme guanyl transferase. This enzyme catalyzes the reaction between the 5'
end of the RNA transcript and a guanine triphosphate (GTP) molecule. Capping involves the addition of a guanine nucleotide
(generally 7-methyl guanosine) to the terminal 5’ nucleotide by an unusual
5’-5’ linkage and the addition of two methyl groups to the first two
nucleotides of the RNA chain. Capping
occurs when the transcript is 20-30 nucleotides long. The process is carried out by a capping
enzyme.
·
5' Terminal phosphate
group is removed by RNA triphosphatase, leaving a bisphosphate group
·
GTP is added to the
terminal bisphosphate by mRNA guanylyl transferase
·
The 7-nitrogen of guanine
is methylated by mRNA (guanine-N7-)-methyltransferase, using S-adenosyl-L-methionine
·
The ribose of the
adjacent 1-2 nucleotides will be methylated to give a cap 1.
The
cap is essential for the ribosome to bind to the 5’ end of mRNA during
translation and it protects the 5' end of the primary RNA transcript from
attack by ribonucleases that have specificity to the 3'5' phosphodiester bonds.
2. Addition
of 3’ poly(A) tail: Poly(A) polymerase adds 150-200 ‘A’ residues are added to
the 3’ end of the mRNA. The poly(A) tail increases the stability of the mRNA in
eukaryotes.
3. Splicing:
The primary transcripts often contain intervening sequences (introns) that are
removed from the RNA prior to translation by a cleavage reaction catalyzed by
snRNPs (small nuclear ribonuclear proteins which contain RNA and protein).
Frequently, the splicing site in the intron has a GU at the 5’ end and an AG at
the 3’ end. The snRNP aligns these ends in a lariat formation to allow precise
splicing.
Splicing
is important since
(1)
Splicing allows variations of a gene and therefore gene product to be made
(2)
It has been suggested that exons correspond to functional motifs in proteins
and thus the presence of genes that require slicing allows for evolutionary
tinkering (attempt to repair or improve)
(3)
Many viruses have spliced mRNAs and so understanding the process may lead to
new therapeutic approaches.
RNA export: RNA synthesis and processing occurs in the nucleus. The mature mRNA is then transported through the nuclear pores in the nuclear envelope to the cytoplasm.
|
Prokaryotic
transcription |
Eukaryotic
transcription |
|
|
Transcription and translation are coupled |
Not Coupled |
|
|
1 |
Occurs in the cytoplasm. |
Occurs in the nucleus. |
|
2 |
RNAs are released and
processed in the cytoplasm. |
RNAs are released and
processes in the nucleus. |
|
3 |
Pre RNA molecules are
released and processed in the cytoplasm. |
Pre RNA are released and
processed in the nucleus. |
|
4 |
A single RNA polymerase |
RNA polymerases I, II and III
|
|
5 |
RNA polymerases are complexes
of five polypeptides. |
RNA polymerases are complexes
of 10-15 polypeptides. |
|
6 |
Initiation of transcription
does not need any proteins or initiation factors. |
Initiation of transcription
requires proteins called transcription factors. |
|
7 |
There is no definite phase
for its occurrence. |
Take place in the G1 and G2
phases of cell cycle. |
|
9 |
The mRNA primary transcript
has fewer additional nucleotides. |
The mRNA primary transcript
has a large number of additional nucleotides. |
|
10 |
Transcriptional unit has one
or more genes (Polycistronic). |
Transcriptional unit has only
one gene (Monocistronic). |
|
11. |
Transcription and translation nearly simultaneous. Little
process of mRNA |
Processing of hnRNA includes Addition of 5’cap and Addition of
3’poly A tail and splicing |
|
12 |
The 23S, 16S and 5S rRNAs are
formed from a single primary transcript. |
The 28S, 18S, 5.8S and 5S rRNAS are formed from two primary
transcripts. |
|
13. |
Inhibitors: Rifampin: RNA polymerase
binds to β subunit. Actinomycin-Intercalates to
interrupt transcription. |
Inhibitors: α amanitin: Inhibits RNA polymerase 2 most srongly |
Translation
Translation is the production of a
polypeptide (protein) using RNA as a template and tRNA molecules as “adapters”
that convert the nucleic acid code to protein code. In translation, messenger RNA (mRNA)—produced
by transcription from DNA is decoded by a ribosome to produce a specific amino
acid chain, or polypeptide. The polypeptide later folds into an active protein.
The ribosome facilitates decoding by inducing the binding of complementary tRNA
anticodon sequences to mRNA codons. The tRNAs carry specific amino acids. The aminoacids carried by t RNA are chained
together into a polypeptide as the mRNA passes through and is "read"
by the ribosome.
There are about 45 different kinds of tRNA molecules. Particular amino acids become associated with particular tRNA molecules by activating enzymes called aminoacyl-tRNA synthetases. There are 20 aminoacyl-tRNA synthetases each recognizing 1 amino acid and the tRNAs that to which that amino acid is to be attached. The enzyme catalyze the bonding between specific tRNAs and the amino acids and the product of this reaction is an aminoacyl-tRNA
Factors
required for Prokaryotic translation
1. mRNA
– contains a RBS (ribosome binding site ) / also known as a Shine- Delgarno
sequence. The Shine-Dalgarno (SD) sequence is a ribosomal binding site in
prokaryotic mRNA, generally located around 8 bases upstream of the start codon
AUG. The RNA sequence helps to recruit the ribosome to the mRNA to initiate
protein synthesis by aligning the ribosome with the start codon. The RBS is
characterized by a core sequence 5’AGGAGU3’.
2. tRNA
– is a type of RNA molecule that helps decode a messenger RNA (mRNA) sequence
into a protein.
a)
anticodon - a 3 nucleotide sequence that is complementary and antiparallel to
mRNA codon
b)
amino acid attachment site at the 3’ end for attachment of amino acid
Aminoacyl tRNA = tRNA with amino acid
attached = charged tRNA.
3. Aminoacyl
tRNA synthetase – transfers the amino acid to its proper tRNA; there are 20 of these,
each recognizing 1 amino acid and the corresponding tRNAs.
4. Ribosomes:
A ribosome is made from complexes of RNAs and proteins and is a
ribonucleoprotein. Each ribosome is divided into two subunits: a smaller
subunit which binds to a larger subunit and the mRNA pattern, and a larger
subunit which binds to the tRNA, the amino acids, and the smaller subunit.
a)
Large subunit (50S) – consists of 23S and 5S rRNAs and 31 ribosomal proteins
b)
Small subunit (30S) - consists of 16S rRNA and 21 ribosomal proteins
The ribosome has three sites for tRNA
to bind. They are the aminoacyl site (abbreviated A), the peptidyl
site (abbreviated P) and the exit site (abbreviated E).
The A site binds the incoming tRNA
with the complementary codon on the mRNA.
The P site holds the tRNA with the
growing polypeptide chain.
The E site holds the tRNA without its
amino acid.
When an aminoacyl-tRNA initially
binds to its corresponding codon on the mRNA, it is in the A site. Then, a
peptide bond forms between the amino acid of the tRNA in the A site and the
amino acid of the tRNA in the P site. The growing polypeptide chain is
transferred to the tRNA in the A site from the tRNA in the P site.
Translocation occurs, moving the tRNA in the P site (without an amino acid) to
the E site. the tRNA that was in the A site, now charged with the polypeptide
chain, is moved to the P site. The tRNA in the E site leaves and another
aminoacyl-tRNA enters the A site to repeat the process.
5. Translation factors
a) Initiation factors
(1) IF1 – promotes association of ribosomal subunits
(2) IF2 (·GTP) – required for fMET-tRNA binding
(3) IF3 – required for mRNA binding, finding the AUG
b) Elongation factors
(1) EF-Tu (·GTP) – binds aminoacid-tRNA to the
ribosome
(2) EF-Ts – regenerates EF-Tu·GTP
(3) EF-G (·GTP) – increases translocation rate
c) Termination factors
(1) RF1 – recognizes UAA and UAG stop codons
(2) RF2 – recognizes UAA and UGA nonsense codons
(3) RF3 (·GTP) – enhanced RF-1 and –2 binding to
ribosome
6. Amino
acids
7. F-met
(N-formyl Met-tRNA)
8. GTP
9. ATP
(for charging tRNAs)
Initiation
In prokaryotes, polypeptide synthesis
begins with the formation of initiation complex.
1. IF1,
IF2·GTP, and IF3 bind to the 30S subunit.
2. Binding
of mRNA and fmet-tRNA to the 30S subunit facilitated by IF3. fmet-tRNA is hydrogen bonded to the AUG
initiation codon in the P site. This forms the 30S initiation complex. Release
of IF3 occurs.
3. 50S
subunit binds with GTP hydrolysis and release of IF1 and IF2. This is the 70S initiation complex.
IF1, IF2 and IF3 are recycled for further uses. fmet-tRNA is bound to mRNA in the P site.
Elongation
Addition of amino
acids to the growing polypeptide chain involves three steps
1. Binding
of aminoacyl tRNA
2. Peptide
Bond formation
3. Translocation
Binding
of aminoacyl tRNA: at the start of
elongation phase, fmet-tRNA is bound to mRNA in the P site in the 70S
initiation complex. An appropriate
aminoacyl tRNA binds to the mRNA codon in the A site. This is done with the help of elongation
factor EF-Tu and GTP. GTP gets hydrolysed
and EF-Tu –GDP is released.
EF-Tu is recycled
by using elongation factor EF-Ts. EF-Ts
bind to EF-Tu –GDP and displace GDP.
EF-Ts -EF-Tu complex binds to GTP and EF-Ts is released regenerating
EF-Tu-GTP.
Peptide
Bond formation: Now the fmet-tRNA is bound to mRNA in the P site and aminoacyl tRNA is
bound in the A site. This position
allows the formation of peptide bond between the two amino acids. Initially the bond between fMet and tRNA is
broken and the enzyme peptidyl transferase catalyses the peptide bond formation
between fMet and the amino acid in the aminoacyl tRNA. Now the tRNA without any
amino acid is left in the P site. The
tRNA in the A site carries the newly formed peptide.
Translocation:
with the tRNA without any amino acid is
left in the P site and the newly formed peptidyl tRNA in the A site, the
ribosome moves one codon along the mRNA toward the 3’ end. This translocation is by the activity of elongation
factors EF-G. EF-G-GTP complex bind to
ribosome and the hydrolysis of GTP the ribosome to move. This results in positioning the peptidyl tRNA
in the P site and empty tRNA in the E site.
The A site is vacant and next aminoacyl tRNA complementary to the mRNA
codon can bind here.
The three steps are repeated until the translation
terminates at the stop codon.
Termination
Elongation continues until the polypeptide
coded by the mRNA is completed. The end
of polypeptide chain is signalled by one of the three stop codons, UAA, UAG and
UGA. Ribosome recognises the termination
codon with the help of termination factors or release factors, RF1, RF2 and
RF3. RF1 recognises UAA and UAG while
RF2 recognises UAA and UGA. RF3
stimulates the termination events.
1. The
release of poly peptide from the tRNA in the P site
2. Release
of the tRNA from the ribosome
3. Dissociation of two ribosome subunits from mRNA
Eukaryotic Translation is similar to prokaryotes except
|
|
Prokaryotes |
Eukaryotes |
|
1 |
It occurs on 70 S ribosomes. |
It occurs on 80 S ribosomes. a) Large subunit (60S)– consists of 28S, 5.8S, and
5S rRNAs and 50 ribosomal proteins b) Small subunit (40S)- consists of 18S rRNA and 33
ribosomal proteins |
|
2 |
It is a continuous process as both
transcription and translation occur in cytoplasm. |
It is a discontinuous process as
transcription occurs in nucleus while translation takes place in cytoplasm. |
|
3 |
mRNA is polycistronic. Bacteria often include several genes within
a single mRNA transcript (polycistronic mRNA), |
mRNA is monicistronic. Each eukaryotic gene is transcribed on a
separate mRNA (monocistronic mRNA). |
|
4 |
First amino acid taking part is
fmet. |
First amino acid is met
(methionine). |
|
5 |
Initiation codon is usually AUG,
occasionally GUG or UUG. |
Initiation codon is AUG.
occasionally GUG or CUG. |
|
6 |
It is a faster process, adds about
20 amino acids per second. |
It is a slower process that adds
one amino acid per second, thus a slower process. |
|
7 |
It requires 3 initiation factors
IFI. IF2. IF3. |
It requires a set of 9 initiation
factors elF 1, 2, 3, 4A. 4B, 4C, 4D, 5. 6. |
|
8 |
After translation, formyl group
from first formylated methionine is removed, retaining methionine in the
polypeptide chain. |
The whole of initiating methionine
is removed from the polypeptide chain. |
|
9 |
It requires two release factors RF1
(for UAG and UAA) and RF2 (for UAA and UGA) in the termination. |
It requires single release factor
eRF 1. |
|
10 |
mRNA life is short (some seconds to some minutes) as
mRNA is less stable. |
mRNA has a life of few hours to few days. It is
quite stable |
Polysome : A polyribosome (or polysome) is a complex of an mRNA molecule and two or more
ribosomes that is formed during active translation.
Many ribosomes simultaneously read one mRNA progressing along the mRNA to
synthesize the same protein.
Post-translational modification
Post-translational modification (PTM) refers to the covalent and generally enzymatic modification of proteins during or after protein biosynthesis. Proteins are synthesized by ribosomes translating mRNA into polypeptide chains, which may then undergo PTM to form the mature protein product. The process of gene expression is not finished when an mRNA has been translated. Many post translational modifications may be required for a polypeptide to fold into the shape required for function.
Prokaryotic organisms generally lack
post-translational modifications. In
eukaryotes there are a number of post-translational modifications after protein
synthesis.
Amino-terminal
modifications of polypeptides: These are the most
common processing events and occur on most cytosolic proteins. Two types of
events normally occur – removal of the N-terminal Met residue, catalyzed by Met
aminopeptidase (MAP) and acetylation of the N – terminal residue, catalysed by
N-acetyltransferase (NAT). Both enzymes are associated with ribosomes and act
on nascent polypeptide.
Example - N-acetylation of melanocyte-stimulating hormone
is required for full biological activity.
Disulphide bond formation:
In eukaryotes, formation of disulphide bond (cys-s-s-cys) occurs in the lumen
of RER and is mediated by an enzyme called disulphide isomerase. This is important in stabilization of
tertiary structure. An improperly folded protein is unstable and lacks
activity.
Disulphide bond is formed in
secretory proteins and exoplasmic membrane proteins.
Modification
of proteins by SUMO: There are Modification
of proteins by SUMO (small ubiquitin-related modifier) addition
Ubiquitin
and Targeted Protein Degradation: A polypeptide
begins to fold as soon as it leaves the ribosome. Some polypeptides can fold into their
complete, mature conformation without help. However, most polypeptides required
chaperones to help them fold properly. Misfolded
proteins are destroyed by Proteosomes. The
proteosome is a barrelshaped, multisubunit protease. Misfolded proteins enter
one end and come out the other as small chains of amino acids (peptides) that are
ultimately recycled. Ubiquitin binds to
misfolded proteins, targeting them for destruction by the proteosome.
Proteolytic cleavages:
Proteolytic cleavage of a precursor form is required in some cases. Selected
segments of amino acid sequences are removed to yield a functional protein.
Glycosylation:
This posttranslational modification has important function in secretion,
antigenicity and clearance of glycoproteins.
Oligosaccharides can be attached to
proteins in three ways:
(i) Via an N-glycosidic bond to the
R-group of an Asn-residue within the consensus sequence Asn-X-Ser/ Thr (N-glycosylation).
(ii) Via an O-gIycosidic bond to the
R group of the Ser or Thr (O-glycosylation). O-linked glycosylation is
extensive in structural proteins such as proteoglycans.
(iii) Carbohydrates are also
components of the glycophosphotidylinositol anchor used to secure some proteins
to the cell membrane.
Glycosylation is both organism- and
cell type-specific and affect the function and immunogenicity of the protein.
Modification of amino
acid within proteins: Modifications of this
type include phosphorylation, acetylation, sulphation, acylation
(carboxylation, myristylation and palmitylation).
Modifications may include:
·
Phosphorylation, the
addition of a phospahate group
·
Methylation, the addition
of a methyl group
·
Subunit binding to form a
multisubunit protein
·
Acylation, In most cases
the initiator methionine is hydrolyzed and an acetyl group is added to the new
N-terminal amino acid.
·
Sulfation, Sulfate modification
of proteins occurs at tyrosine residues. As many as 1% of all tyrosine residues
present in the eukaryotic proteome are modified by sulfate addition
·
Prenylation, addition of
the 15 carbon farnesyl group or the 20 carbon geranylgeranyl group
·
Vitamin C-Dependent
Modifications, Modifications of proteins that depend upon vitamin C as a
cofactor include proline and lysine hydroxylations and carboxy terminal
amidation.
·
Vitamin K-Dependent
Modifications, carboxylation of glutamic acid residues
·
Selenoproteins,
Selenocysteine incorporation in eukaryotic proteins
Plasma membrane proteins and secreted
proteins are post-translationally modified in the Rough Endoplasmic Reticulum
(RER) and the Golgi.
Purposes
of Post-translational Events & Modifications:
·
Quality Control: Chaperones, Glycosylation
·
Degradation of misfolded
proteins: Ubiquitination
·
Proper protein function:
Glycosylation, Phosphorylation, Ubiquitination
·
Target protein to proper
locations: Acylation, GPI anchors
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