2. TRANSLATION IN
BACTERIA
Initiation:
Initiation of translation in prokaryotes involves the
assembly of the components of the translation system, which are:
the two ribosomal subunits (50S and 30Ssubunits); the mature
mRNA to be translated; the tRNA charged with N-
formylmethionine (the first amino acid in the nascent
peptide); guanosine triphosphate(GTP) as a source of energy; the
prokaryotic elongation factor EF-P and the three prokaryotic
initiation factors IF1, IF2, and IF3, which help the assembly of the
initiation complex. Variations in the mechanism can be
anticipated.
The ribosome has three active sites: the A site, the P
site, and the E site. The A site is the point of entry for the
aminoacyl tRNA (except for the first aminoacyl tRNA, which
enters at the P site). The P site is where the peptidyl tRNA is
formed in the ribosome. And the E site which is the exit site of the
now uncharged tRNA after it gives its amino acid to the growing
peptide chain.
The selection of an initiation site (usually an AUG
codon) depends on the interaction between the 30S subunit and
the mRNA template. The 30S subunit binds to the mRNA
template at a purine-rich region (the Shine-Dalgarno sequence)
upstream of the AUG initiation codon. The Shine-Dalgarno
sequence is complementary to a pyrimidine rich region on the
16S rRNA component of the 30S subunit. During the formation of
3. the initiation complex, these complementary nucleotide
sequences pair to form a double stranded RNA structure that
binds the mRNA to the ribosome in such a way that the initiation
codon is placed at the P site.
5. Elongation of the polypeptide chain involves
addition of amino acids to the carboxyl end of the growing chain.
The growing protein exits the ribosome through the polypeptide
exit tunnel in the large subunit.
Elongation starts when the fMet-tRNA enters the P site,
causing a conformational change which opens the A site for the
new aminoacyl-tRNA to bind. This binding is facilitated
by elongation factor-Tu (EF-Tu), a small GTPase. For fast and
accurate recognition of the appropriate tRNA, the ribosome
utilizes large conformational changes (conformational
proofreading). Now the P site contains the beginning of the
peptide chain of the protein to be encoded and the A site has the
next amino acid to be added to the peptide chain. The growing
polypeptide connected to the tRNA in the P site is detached from
the tRNA in the P site and a peptide bond is formed between the
last amino acids of the polypeptide and the amino acid still
attached to the tRNA in the A site. This process, known
as peptide bond formation, is catalyzed by a ribozyme (the 23S
ribosomal RNA in the 50S ribosomal subunit). Now, the A site has
the newly formed peptide, while the P site has an uncharged
tRNA (tRNA with no amino acids). The newly formed peptide in
the A site tRNA is known as dipeptide and the whole assembly is
called dipeptidyl-tRNA. The tRNA in the P site minus the amino
acid is known to be deacylated. In the final stage of elongation,
called translocation, the deacylated tRNA (in the P site) and
thedipeptidyl-tRNA (in the A site) along with its corresponding
codons move to the E and P sites, respectively, and a new codon
moves into the A site. This process is catalyzed by elongation
factor G (EF-G). The deacylated tRNA at the E site is released
from the ribosome during the next A-site occupation by an
aminoacyl-tRNA again facilitated by EF-Tu.
The ribosome continues to translate the remaining
codons on the mRNA as more aminoacyl-tRNA bind to the A site,
6. until the ribosome reaches a stop codon on mRNA(UAA, UGA, or
UAG).
The translation machinery works relatively slowly compared to the
enzyme systems that catalyze DNA replication. Proteins in
prokaryotes are synthesized at a rate of only 18 amino acid
residues per second, whereas bacterial replisomes synthesize
DNA at a rate of 1000 nucleotides per second. This difference in
rate reflects, in part, the difference between polymerizing four
types of nucleotides to make nucleic acids and polymerizing 20
types of amino acids to make proteins. Testing and rejecting
incorrect aminoacyl-tRNA molecules takes time and slows protein
synthesis. In bacteria, translation initiation occurs as soon as the
5' end of an mRNA is synthesized, and translation and
transcription are coupled. This is not possible in eukaryotes
because transcription and translation are carried out in separate
compartments of the cell (the nucleus and cytoplasm).
Termination:
Termination occurs when one of the three termination
codons moves into the A site. These codons are not recognized
by any tRNAs. Instead, they are recognized by proteins
7. called release factors, namely RF1 (recognizing the UAA and
UAG stop codons) or RF2 (recognizing the UAA and UGA stop
codons). These factors trigger the hydrolysis of the ester bond in
peptidyl-tRNA and the release of the newly synthesized protein
from the ribosome. A third release factor RF-3 catalyzes the
release of RF-1 and RF-2 at the end of the termination process.
Recycling:
The post-termination complex formed by the end of
the termination step consists of mRNA with the termination codon
at the A-site, an uncharged tRNA in the P site, and the intact 70S
ribosome. Ribosome recycling step is responsible for the
disassembly of the post-termination ribosomal complex. Once the
nascent protein is released in termination, Ribosome Recycling
Factor and Elongation Factor G (EF-G) function to release mRNA
and tRNAs from ribosomes and dissociate the 70S ribosome into
the 30S and 50S subunits. IF3 then replaces the deacylated tRNA
releasing the mRNA. All translational components are now free
8. for additional rounds of translation.
Polysomes:
Translation is carried out by more than one
ribosome simultaneously. Because of the relatively large size of
ribosomes, they can only attach to sites on mRNA 35 nucleotides
apart. The complex of one mRNA and a number of ribosomes is
9. called a polysome or polyribosome
Regulation of translation:
When bacterial cells run out of nutrients, they
enter stationary phase and down regulate protein synthesis.
Several processes mediate this transition. For instance, in E. coli,
70S ribosomes form 90S dimers upon binding with a small 6.5
kDa protein, ribosome modulation factor RMF. These
intermediate ribosome dimers can subsequently bind
a hibernation promotion factor protein, HPFmolecule to form a
mature 100S ribosomal particle, in which the dimerization
interface is made by the two 30S subunits of the two participating
ribosomes. The ribosome dimers represent a hibernation state
and are translationally inactive. A third protein that can bind to
ribosomes when E. coli cells enter the stationary phase
is YfiA (previously known as RaiA). HPF and YfiA are structurally
similar, and both proteins can bind to the catalytic A- and P-sites
of the ribosome. RMF blocks ribosome binding to mRNA by
preventing interaction of the messenger with 16S rRNA.When
bound to the ribosomes the C-terminal tail of E. coli YfiA interferes
10. with the binding of RMF, thus preventing dimerization and
resulting in the formation of translationally inactive monomeric
70S ribosomes.
Mechanism of ribosomal subunit dissociation by RsfS (= RsfA)
In addition to ribosome dimerization, the joining of the two
ribosomal subunits can be blocked by RsfS (formerly called RsfA
or YbeB). RsfS binds to L14, a protein of the large ribosomal
subunit, and thereby blocks joining of the small subunit to form a
functional 70S ribosome, slowing down or blocking translation
entirely. RsfS proteins are found in almost all eubacteria (but
not archaea) and homologs are present
in mitochondria and chloroplasts . However, it is not known yet
how the expression or activity of RsfS is regulated.
Effect of antibiotics:
Several antibiotics exert their action by
targeting the translation process in bacteria. They exploit the
differences to selectively inhibit protein synthesis in bacteria
11. without affecting between prokaryotic and eukaryotic
translationmechanis the host.
Microbial protein production in bacteria
Special vectors for expression of foreign genes
in E.coli:
If a foreign (i.e., non-bacterial) gene is simply ligated into a standard
vector and cloned in E. coli, it is very unlikely that a significant amount
of recombinant protein will be synthesized. This is because expression
is dependent on the gene being surrounded by a collection of signals
that can be recognized by the bacterium. These signals, which are short
sequences of nucleotides, advertise the presence of the gene and
provide instructions for the transcriptional and translational apparatus
of the cell. The three most important signals for E. coli genes are as
follows the promoter, which marks the point at which transcription of
12. the gene should start. In E. coli, the promoter is recognized by the
subunit of the transcribing enzyme RNA polymerase.
The terminator, which marks the point at the end of the gene where
transcription should stop. A terminator is usually a nucleotide sequence
that can base pair with itself to form a stem–loop structure.
The ribosome binding site, a short nucleotide sequence recognized by
the ribosome as the point at which it should attach to the mRNA
molecule. The initiation codon of the gene is always a few nucleotides
downstream of this site.
The genes of higher organisms are also surrounded by expression
signals, but their nucleotide sequences are not the same as theE. coli
versions. This is illustrated by comparing the promoters of E. coli and
human genes . There are similarities,but it is unlikely that an E. coli RNA
polymerase would be able to attach to a human promoter. A foreign
gene is inactive in E. coli, simply because the bacterium does not
recognize its expression signals. A solution to this problem would be to
13. insert the foreign gene into the vector in such a way that it is placed
under control of a set of E. coli expression signals. If this can be
achieved, then the gene should be transcribed and translated . Cloning
vectors that provide these signals, and can therefore be used in the
production of recombinant protein, are called expression vectors.
The promoter is the critical component of an expression
vector
The promoter is the most important component of an
expression vector. This is because the promoter controls the very first
stage of gene expression (attachment of an RNA polymerase enzyme to
the DNA) and determines the rate at which mRNA is synthesized. The
amount of recombinant protein obtained therefore depends to a great
extent on the nature of the promoter carried by the expression vector.
The promoter must be chosen with care. The two sequences shown in
14. are consensus sequences, averages of all the E. coli promoter
sequences that are known. Although most E. coli promoters do not
differ much from these consensus sequences (e.g., TTTACA instead of
TTGACA), a small variation may have a major effect on the efficiency
with which the promoter can direct transcription. Strong promoters are
those that can sustain a high rate of transcription;
strong promoters usually control genes whose translation products are
required in large amounts by the cell . In contrast, weak promoters,
which are relatively inefficient, direct transcription of genes whose
products are needed in only small amounts . Clearly an expression
vector should carry a strong promoter, so that the cloned gene is
transcribed at the highest possible rate.A second factor to be
considered when constructing an expression vector is whether
it will be possible to regulate the promoter in any way. Two major types
of gene regulation are recognized in E. coli—induction and repression.
An inducible gene is one whose transcription is switched on by addition
of a chemical to the growth medium; Often this chemical is one of the
substrates for the enzyme coded by the inducible gene. In contrast, a
repressible gene is switched off by addition of the regulatory chemical
.Gene regulation is a complex process that only indirectly involves the
promoter itself.However, many of the sequences important for
induction and repression lie in the region surrounding the promoter
and are therefore also present in an expression vector.It may therefore
be possible to extend the regulation to the expression vector, so that
the chemical that induces or represses the gene normally controlled by
the promoter is also able to regulate expression of the cloned gene.
This can be a distinct advantage in the production of recombinant
protein.
Examples of promoters used in expression vectors
15. Several E. coli promoters combine the desired features of strength and
ease of regulation.
Those most frequently used in expression vectors are as follows:
l The lac promoter is the sequence that controls transcription of
the lacZ gene coding for b-galactosidase (and also the lacZ′ gene
fragment carried by the pUC and M13mp vectors; p. 79). The lac
promoter is induced by isopropylthiogalactoside (IPTG, p. 80), so
addition of this chemical into the growth medium switches on
transcription of a gene inserted downstream of the lac promoter
carried by an expression vector.
The trp promoter is normally upstream of the cluster of genes coding
for several of the enzymes involved in biosynthesis of the amino acid
tryptophan. The trp promoter is repressed by tryptophan, but is more
easily induced by 3-b-indoleacrylic acid.
l The tac promoter is a hybrid between the trp and lac promoters.
It is stronger than either, but still induced by IPTG.
The EPL promoter is one of the promoters responsible for transcription
of the e DNA molecule. ePL is a very strong promoter that is recognized
by the E. coli RNA polymerase, which is subverted by e into transcribing
the bacteriophage DNA. The promoter is repressed by the product of
the ecI gene. Expression vectors that carry the ePL promoter are used
with a mutant E. coli host that synthesizes a temperature-sensitive
form of the cI protein (p. 40). At a low temperature (less than 30°C) this
mutant cI protein is able to repress the ePL promoter, but at higher
temperatures the protein is inactivated, resulting in transcription of the
cloned gene.
Cassettes and gene fusions
An efficient expression vector requires not only
a strong, regulatable promoter, but also an E. coli ribosome binding
sequence and a terminator. In most vectors these expression signals
form a cassette, so-called because the foreign gene is inserted into a
unique restriction site present in the middle of the expression signal
16. clusters. Ligation of the foreign gene into the cassette therefore places
it in the ideal position relative to the expression signals.
With some cassette vectors the cloning site is not immediately adjacent
to the ribosome binding sequence, but instead is preceded by a
segment from the beginning of an E.coli gene. Insertion of the foreign
gene into this restriction site must be performed in such a way as to
fuse the two reading frames, producing a hybrid gene that starts with
the E. coli segment and progresses without a break into the codons
of the foreign gene. The product of gene expression is therefore a
hybrid or fusion protein, consisting of the short peptide coded by the E.
coli reading frame fused to the amino-terminus of the foreign protein.
This fusion system has four advantages
Efficient translation of the mRNA produced from the cloned gene
depends not only on the presence of a ribosome binding site, but is also
affected by the nucleotide sequence at the start of the coding region.
This is probably because secondary structures resulting from
intrastrand base pairs could interfere with attachment of the ribosome
to its binding site.
General problems with the production of recombinant proteins in
E.coli
Despite the development of sophisticated expression vectors,
there are still numerous difficulties associated with the production
of protein E.coli. These problems can be grouped into two
categories: those that are due to the sequence of the foreign
17. gene, and those that are due to the limitations of E. coli as a host
for recombinant protein synthesis.
Problems resulting from the sequence of foreign gene
The foreign gene might contain introns. This would be a major
problem, as E. coli genes do not contain introns and therefore the
bacterium does not possess the necessary machinery for
removing introns from transcripts. The foreign gene might contain
sequences that act as termination signals in E. coli.These
sequences are perfectly innocuous in the normal host cell,but in
the bacterium result in premature termination and a loss of gene
expression
Problems caused by E. coli
E. coli might not process the recombinant protein correctly. The
proteins of most organisms are processed after translation by
chemical modification of amino acids within the polypeptide. Often
these processing events are essential for the correctbiological
activity of the protein. Unfortunately, the proteins of bacteria and
higher organisms are not processed identically. In particular,
some animal proteins are glycosylated, meaning that they have
sugar groups attached to them after translation. Glycosylation is
extremely uncommon in bacteria and recombinant glycosylated
correctly. E. coli might not fold the recombinant protein correctly,
and generally is unable to synthesize the disulphide bonds
present in many animal proteins. If the protein does not take up its
correctly folded tertiary structure, then usually it is insoluble and
forms an inclusion body within the bacterium. Recovery of the
protein from the inclusion body is not a problem, but converting
the protein into its correctly folded form can be difficult or
18. impossible in the test tube. Under these circumstances the protein
is, of course, inactive. E. coli might degrade the recombinant
protein. Exactly how E. coli can recognize the foreign protein, and
thereby subject it to preferential turnover, is not known.
