Download E.Coli Biological Background of Translation Initiation and

Biological Background of
Translation Process in E.Coli
•
Translation usually initiates at the AUG codon nearest
to the 5´ end of the mRNA molecule. However, this
does not happen in all cases.
•
There are some escape mechanisms that allow the
initiation of translation at following, but still near the 5´
end, AUG codons:
(1) leaky scanning: where the first AUG is bypassed due to
inappropriate context.
(2) Reinitiation, where translation initiates at an AUG codon before
the correct initiation site and ends by reaching a stop codon.
Translation reinitiates when the true AUG codon is found.
(3) direct internal initiation: In this case the ribosome directly
attaches near the true AUG codon without any scanning.
•
These mechanisms of the translation initiation process make
more difficult the recognition of the TIS on a given genomic
sequence.
•
There are three different ways to read a given
sequence in a given direction. Each of these ways of
reading is referred to as reading frame.
(1) The first reading frame starts at position 1,
(2) The second starts at position 2,
(3) The third starts at position 3.
•
The reading frame that is translated into a protein is
named Open Reading Frame (ORF). A codon that is
contained in the same reading frame with respect to
another codon is referred to as “in-frame codon”. The
coding region of an ORF is bounded by the initiation
codon and the first in-frame stop codon. The coding
region is surrounded by non-coding regions called 5´
and 3´ untranslated regions (UTRs).
•
The translation initiation region (TIR) in E.coli mRNA is
characterized by the start codon and the Shine-Dalgarno
base pairing of a region upstream of the gene's coding
sequence with the 3'end of the 16SrRNA.
•
However, these domains are not sufficient to define an
efficient TIR. Additional sequences and structures are
required. How translation is affected by mRNA sequences
upstream of the Shine- Dalgarno region or downstream of
the start codon is not quite clear.
•
A nonrandom distribution of nucleotides in this region,
revealed by statistical analysis, indicates that these
sequences carry additional information in their primary
structure for the efficiency of the initiation signals. Some
scientists’ finding indicates that, nucleotides + 15 to +26, a
sequence complementary to nucleotides 1471 to 1482 of the
16SrRNA, suggesting a second mRNA-rRNA base pairing
contact besides the Shine-Dalgarno interaction.
If you are interested in this base pairing mechanism,
please go to this website:
http://www.pubmedcentral.nih.gov/picrender.fcgi?
artid=330588&blobtype=pdf
I think it could also become a target of your research.
As for the initiation site prediction, I found some
examples, such as this article named “Prediction of
Translation Initiation Sites on the Genome of
Synechocystis sp. Strain PCC6803 by Hidden Markov
Model”.
http://dnaresearch.oxfordjournals.org/cgi/content/abstrac
t/4/3/179
Since I’m not that familiar with computer programming,
this could only serve as a reference.
Translation Factors:
•
Initiation of translation begins with the 50s and 30s
ribosomal subunits dissociated.
1. IF1 (initiation factor 1) blocks the A site to insure that the
fMet-tRNA can bind only to the P site and that no other
aminoacyl-tRNA can bind in the A site during initiation,
2. IF-2 is a small GTPase which binds fmet-tRNA and helps
its binding with the small ribosomal subunit.
3. IF3 blocks the E site and prevents the two subunits from
associating.
•
The 16s rRNA of the small 30S ribosomal subunit
recognizes the ribosomal binding site on mRNA (the
Shine-Dalgarno sequence, 5-10 base pairs upstream of
the start codon(AUG)) The Shine-Delgarno sequence is
found only in prokaryotes. This helps to correctly position
the ribosome onto the mRNA so that the P site is directly
on the AUG initiation codon.
•
IF-3 helps to position fmet-tRNA into the P site, such that
fmet-tRNA interacts via base pairing with the mRNA
initiation codon (AUG). Initiation ends as the large
ribosomal subunit joins the complex causing the
dissociation of initiation factors.
•
Prokaryotes （E. coli） can differentiate between a normal
AUG (coding for methionine) and an AUG initiation codon
(coding for formylmethionine and indicating the start of a
new translation process).
•
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 called release factors, namely
1. RF1 (recognizing the UAA and UAG stop codons) or
2. RF2 (recognizing the UAA and UGA stop codons).
3. RF-3 catalyzes the release of RF-1 and RF-2 at the end
of the termination process.
•
These factors trigger the hydrolysis of the ester bond in
peptidyl-tRNA and the release of the newly synthesized
protein from the ribosome.
•
Example: If RF2 recognizes the stop codon UGA, translation is
terminated after synthesis of a 25 amino acid peptide (which is
degraded). If frameshifting occurs, the complete sequence of RF2 is
synthesized.
The DNA sequence of the release factor 2 (RF2) gene
revealed a stop codon at position 26 where the open reading
frame switched to the +1 frame. Comparison of amino acid
and mRNA sequences showed that ribosomes can shift
reading frame at codon 25, avoid the stop codon, and
decode the main portion of the message.
Further work showed that this site-specific shift in reading
frame is not just "noise" in translation but remarkably
efficient:
30% of the ribosomes make complete RF-2 and
70% terminate at the zero frame UGA after 25 codons.
The RF-2 frameshift site is a "slippery" codon where the
mRNA slides within the ribosome complex one nucleotide by
breaking codon-anticodon pairing with peptidyl tRNA in the
ribosome P site and re-establishing pairing with an
overlapping codon in the new frame.
In this case tRNA- Leu with anticodon 3'-GAG-5' pairs with
CUU in the first frame and then with UUU in the +1 frame
(CUU.UGA), inserting one leucine for four nucleotides in the
mRNA.
Competition between termination and frameshifting is over
which process captures the U of the UGA sequence. Release
factor 2, the recoding product, promotes ribosome termination
at UGA (and UAA) and so competes with recoding by
enhancing termination at UGA, the 26th codon in the original
frame.
Competition is tilted in favor of recoding in two ways, The
presence of a C 3' of the UGA makes a poor termination
context, and CUU is a particularly shift-prone codon.
If extra RF2 is present, frameshifting decreases so that less
RF2 is made.
When RF2 is in short supply termination loses, frameshifting
wins, and RF2 concentration is restored.
The stimulatory signal is a short sequence three nucleotides 5'
of the shift site that pairs with 16S rRNA of the translating
ribosome, just like the Shine-Dalgarno pairing that occurs 5' of
the AUG start codon during ribosome initiation.
Thus, RF-2 mRNA has two Shine-Dalgarno sequences, one
for initiation and one in the coding sequence for promoting
frameshifting.
Spacing between the Shine-Dalgarno sequence and the site
of action is crucial and varies for these cases. With initiation
the optimal spacing to the AUG is 5 bases, although there is
considerable latitude (3 to 12 bases). With RF-2 required
spacing is 3 bases. The implication is that pairing between
mRNA and rRNA at these sites distorts the complex to either
put the ribosome in an "initiation mode", a "plus mode" to
force it forward, or a "minus mode" to force it backward.
BTW, I found an interesting article in this address:
http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=eurekah.sectio
n.19054
It indicated the mechnism of those translational factors,
include initiation factors and release factors. For this article, it
seems that these factors are related to the genome
sequence of E. coli, for example, infA gene is responsible for
IF1 synthesis. infA gene is located at 20 min on the E. coli
chromosome. Therefore, in depth research would focus on
these genes respectively, I wonder if you have time to finish
these research before the deadline.