L5-6 Translation in Prokaryotes

L5
Translation in Prokaryotes
GENETICS AND MOLECULAR BIOLOGY

(BTM-104)
M.Sc. Biotechnology
I Sem
Date: 24 th Jan, 2022
Important aspects of protein synthesis:
Codon–anticodon interaction:
The anticodon at one end of the tRNA interacts with a complementary triplet of bases on
the mRNA, the codon, when both are brought together in the cleft of the ribosome. The
interaction is antiparallel in nature.
Some highly purified tRNA molecules were found to interact with more than one codon, and
this ability correlated with the presence of modified nucleosides in the 5’-anticodon position,
particularly inosine.
Wobble hypothesis:
The wobble hypothesis was suggested by Crick to explain the redundancy of the genetic
code.
He realized, by model building, that the 5’-anticodon base was able to undergo more
movement than the other two bases and could thus form nonstandard base pairs as long as
the distances between the ribose units were close to normal.
- No purine–purine or pyrimidine–pyrimidine base pairs are allowed as the ribose distances
would be incorrect.
- No single tRNA could recognize more than three codons, hence, at least 32 tRNAs would
be needed to decode the 61 codons, excluding stop codons.
- tRNAs can recognize either one, two or three codons, depending on their wobble base
(the 5’-anticodon base).
If it is C it will recognize only the codon ending in G.
If it is G, it will recognize the two codons ending in U or C.
If U, which is subsequently modified, it will pair with either A or G.
The wobble nucleoside is never A, as this is converted to inosine (I) which then pairs
with A, C or U.
[Note: Inosine is formed by post-transcriptional processing of adenosine. This is carried out
by anticodon deaminase which converts the 6-amino group to a keto group.]
Ribosome binding site:
In prokaryotic mRNAs there is a conserved sequence 8–13 nt upstream of the first codon to
be translated (the initiation codon).
- It was discovered by Shine and Dalgarno and is a purine-rich sequence usually containing
all or part of the sequence 5’-AGGAGGU-3’.
- This sequence can base-pair with the 3’-end of the 16S rRNA in the small subunit of the
ribosome (5’-ACCUCCU-3’).
- - It is called the ribosome binding site, or Shine–Dalgarno sequence. It is thought to
position the ribosome correctly with respect to the initiation codon.
- In the middle of mRNA, SD sequence may act as pausing signal for translation.
Polysomes:
When a ribosome has begun translating an mRNA molecule and has moved about 70–80 nt
from the initiation codon, a second ribosome can assemble at the ribosome-binding site and
start to translate the mRNA.
- When this second ribosome has moved along, a third can begin and so on.
- Multiple ribosomes on a single mRNA are called polysomes (short for polyribosomes)
and there can be as many as 50 on some mRNAs, although they cannot be positioned closer
than about 80 nt.
Initiator tRNA :
- The first amino acid incorporated into a protein chain is methionine in both prokaryotes
and eukaryotes, though in the former the Met has been modified to N-formylmethionine.
In both types of organisms, the AUG initiation codon is recognized by a special initiator
tRNA.
- The initiator tRNA differs from the one that pairs with AUG codons in the rest of the coding
region.
- The initiator tRNA allows more flexibility in base pairing (wobble) because it lacks the
alkylated A in the anticodon loop and hence it can recognize both AUG and GUG as
initiation codons, the latter occurring occasionally in prokaryotic mRNAs.
- The noninitiator tRNA is less flexible and can only pair with AUG codons.
- Both tRNAs are charged with Met by the same methionyl-tRNA synthetase to give the
methionyl-tRNA, but only the initiator methionyl-tRNA is modified by the enzyme
transformylase to give N-formylmethionyl-tRNA (fMet-tRNA).
- The N-formyl group resembles a peptide bond and may help this initiator tRNA to enter
the P-site of the ribosome whereas all other tRNAs enter the A-site.
The actual mechanism of protein synthesis can be divided into three stages:
● initiation – the assembly of a ribosome on an mRNA molecule;

● elongation – repeated cycles of amino acid addition;
● termination – the release of the new protein chain.
In prokaryotes, the factors are abbreviated as IF or EF for initiation and elongation

factors respectively, whereas in eukaryotes they are called eIF and eEF.
There are distinct differences of detail between the mechanism in prokaryotes and
eukaryotes, and most of these occur in the initiation stage.
Initiation:
The purpose of the initiation step is to assemble a complete ribosome on to an mRNA
molecule at the correct start point, the initiation codon. The components involved are the
large and small ribosome subunits, the mRNA, the initiator tRNA in its charged form,
three initiation factors and GTP.
The overall sequence of events is as follows:
1) IF1 and IF3 bind to a free 30S subunit. This helps to prevent a large subunit
binding to it without an mRNA molecule and forming an inactive ribosome.
2) IF2 complexed with GTP then binds to the small subunit. It will assist the
charged initiator tRNA to bind.
3) The 30S subunit attaches to an mRNA molecule making use of the ribosomebinding
site (RBS) on the mRNA
4) The initiator tRNA can then bind to the complex by base pairing of its anticodon
with the AUG codon on the mRNA. At this point, IF3 can be released,
as its roles in keeping the subunits apart and helping the mRNA to bind are
complete. This complex is called the 30S initiation complex.
5) The 50S subunit can now bind, which displaces IF1 and IF2, and the GTP is
hydrolyzed in this energy-consuming step. The complex formed at the end
of the initiation phase is called the 70S initiation complex.
-The assembled ribosome has two tRNA-binding sites. These are called the A- and P-sites,
for aminoacyl and peptidyl sites.
- The A-site is where incoming aminoacyl-tRNA molecules bind, and the P-site is where
the growing polypeptide chain is usually found. These sites are in the cleft of
the small subunit and contain adjacent codons that are being translated.
- One major outcome of initiation is the placement of the initiator tRNA in the P-site. It is
the only tRNA that does this, as all others must enter the A-site.
Elongation: With the formation of the 70S initiation complex, the elongation cycle can
begin. It can be subdivided into three steps as follows:
(i) aminoacyl-tRNA delivery,
(ii) peptide bond formation and
(iii) translocation (movement)
The process beginning where the P-site is occupied and the A-site is empty.
It involves three elongation factors EF-Tu, EF-Ts and EF-G which all bind GTP or GDP.
(i) Aminoacyl-tRNA delivery.

-EF-Tu is required to deliver the aminoacyl-tRNA to the A-site and energy is consumed in
this
step by the hydrolysis of GTP.
- The released EF-Tu-GDP complex is regenerated with the help
of EF-Ts. In the EF-Tu–EF-Ts exchange cycle, EF-Ts displaces the GDP and subsequently is
displaced itself by GTP.
- The resultant EF-Tu GTP complex is now able to bind another aminoacyl-tRNA and deliver
it to the ribosome.
- - All aminoacyl-tRNAs can form this complex with EF-Tu, except the initiator tRNA.
(ii) Peptide bond formation.
- After aminoacyl-tRNA delivery, the A- and P-sites are both occupied and the two amino
acids that are to be joined are in close proximity.
- The peptidyl transferase activity of the 50S subunit can now form a peptide bond
between these two amino acids without the input of any more energy, since energy in the
form of ATP was used to charge the tRNA.
(iii) Translocation.
- A complex of EF-G (translocase) and GTP binds to the ribosome and, in an energy-
consuming step, the discharged tRNA is ejected from the P-site, the peptidyl-tRNA is moved
from the A-site to the P-site and the mRNA moves by one codon relative to the ribosome.
- GDP and EF-G are released, the latter being re-usable.
- A new codon is now present in the vacant A-site.
- Recent evidence suggests that in prokaryotes the discharged tRNA is first moved to an
E-site (exit site) and is ejected when the next aminoacyl-tRNA binds. In this way the
ribosome maintains contact with the mRNA via 6 base pairs which may well reduce the
chances of frameshifting.
One cycle of the three-step elongation cycle has been completed, and the cycle
is repeated until one of the three termination codons (stop codons) appears in
the A-site.
Termination:
- There are no tRNA species that normally recognize stop codons.
- Instead, protein factors called release factors interact with these codons and cause
release of the completed polypeptide chain.
- - RF1 recognizes the codons UAA and UAG, and RF2 recognizes UAA and UGA. RF3 helps
either RF1 or RF2 to carry out the reaction.
- The release factors make peptidyl transferase transfer the polypeptide to water rather
than to the usual aminoacyl-tRNA, and thus the new protein is released.
- To remove the uncharged tRNA from the P-site and release the mRNA, EF-G together with
ribosome release factor are needed for the complete dissociation of the subunits.
- IF3 can now bind the small subunit to prevent inactive 70S ribosomes forming.

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L5

GENETICS AND MOLECULAR BIOLOGY

● initiation – the assembly of a ribosome on an mRNA molecule;

In prokaryotes, the factors are abbreviated as IF or EF for initiation and elongation

(i) Aminoacyl-tRNA delivery.

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