Protein biosynthesis in prokaryotes: Initiation, elongation, termination.

ATTACHMENT OF AMINO ACIDS TO tRNA

tRNA molecules to which an amino acid is attached are said to be charged, and tRNAs that lack an
amino acid are said to be uncharged. Charging requires an acyl linkage between the carboxyl group
of the amino acid and the 2’- or 3’-hydroxyl group of the adenosine nucleotide that protrudes from
the acceptor stem at the 3’ end of the tRNA. This acyl linkage is a high-energy bond because its
hydrolysis results in a large change in free energy.

All aminoacyl-tRNA synthetases attach an amino acid to a tRNA in two enzymatic steps (Fig. 1).
Step one is adenylylation in which the amino acid reacts with ATP to become adenylylated with the
concomitant release of pyrophosphate. Adenylylation refers to transfer of AMP, as opposed to
adenylation, which would indicate the transfer of adenine. The principal driving force for the
adenylylation reaction is the subsequent hydrolysis of pyrophosphate by pyrophosphatase. As a
result of adenylylation, the amino acid is attached to adenylic acid via a high-energy ester bond in
which the carbonyl group of the amino acid is joined to the phosphoryl group of AMP. Step two is
tRNA charging in which the adenylylated amino acid, which remains tightly bound to the synthetase,
reacts with tRNA. This reaction results in the transfer of the amino acid to the 3’ end of the tRNA via
the 2’- or 3’-hydroxyl and the release of AMP.

There are two classes of tRNA synthetases. Class I enzymes attach the amino acid to the 2’-OH of
the tRNA and are generally monomeric. Class II enzymes attach the amino acid to the 3’-OH of the
tRNA and are typically dimeric or tetrameric. Although the initial coupling between the tRNA and
the amino acid is different, once released from the synthetase, the amino acid rapidly equilibrates
between attachment at the 3’-OH and the 2’-OH.

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Fig. 1 The two steps of aminoacyl-tRNA charging. (a) Adenylylation of amino acid. (b) Transfer of the
adenylylated amino acid to tRNA. The process shown is for a class II tRNA synthetase (which attaches
the amino acid to the 3’-OH). (Source: Molecular Biology of Genes)

MESSENGER RNA
The translation machinery decodes only a portion of each mRNA. The information for protein
synthesis is in the form of three-nucleotide codons, which each specifies one amino acid. The
protein-coding region(s) of each mRNA is composed of a contiguous, non-overlapping string of
codons called an open reading frame (ORF). Each ORF specifies a single protein and starts and ends
at internal sites within the mRNA. That is, the ends of an ORF are distinct from the ends of the
mRNA. Translation starts at the 5’ end of the ORF and proceeds one codon at a time to the 3’ end.
The first and last codons of an ORF are known as the start and stop codons. In bacteria, the start
codon is usually 5’-AUG-3’, but 5’-GUG-3’ and sometimes even 5’-UUG-3’ are also used (rear cases).
Eukaryotic cells always use 5’-AUG-3’ as the start codon. The start codon has two important
functions. First, it specifies the first amino acid to be incorporated into the growing polypeptide
chain. Second, it defines the reading frame for all subsequent codons. Because each codon is
immediately adjacent to (but not overlapping with) the next codon, and because codons are three
nucleotides long, any stretch of mRNA could be translated in three different reading frames. Once

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translation starts, however, the reading frame is determined. Thus, by setting the location of the first
codon, the start codon determines the location of all following codons.

Stop codons, of which there are three (5’-UAG-3’, 5’-UGA-3’, and 5’-UAA-3’), define the end of the
ORF and signal termination of polypeptide synthesis. ORF is a contiguous stretch of codons “read” in
a particular frame (as set by the first codon) that is “open” to translation because it lacks a stop
codon.

mRNAs contain at least one ORF. The number of ORFs per mRNA is different between eukaryotes
and prokaryotes. Eukaryotic mRNAs almost always contain a single ORF. In contrast, prokaryotic
mRNAs frequently contain two or more ORFs and hence can encode multiple polypeptide chains.
mRNAs containing multiple ORFs are known as polycistronic mRNAs, and those encoding a single
ORF are known as monocistronic mRNAs. The polycistronic mRNAs found in bacteria often encode
proteins that perform related functions, such as different steps in the biosynthesis of an amino acid
or nucleotide.

Protein synthesis in prokaryotes:

For translation to occur, the ribosome must be recruited to the mRNA. To facilitate binding by a
ribosome, many prokaryotic ORFs contain a short sequence upstream (on the 5’ side) of the start
codon called the ribosomebinding site (RBS). This element is also referred to as a Shine–Dalgarno
sequence after the scientists who discovered it by comparing the sequences of multiple mRNAs. The
RBS, typically located 3–9 bp on the 5’ side of the start codon, is complementary to a sequence
located near the 3’ end of one of the ribosomal RNA components, the 16S ribosomal RNA (rRNA)
(Fig. 2). The RBS base-pairs with this RNA, there by aligning the ribosome with the beginning of the
ORF. The core of this region of the 16S rRNA has the sequence 5’-CCUCCU-3’. Prokaryotic RBS are
most often a subset of the sequence 5’-AGGAGG-3’. The extent of complementarity and the spacing
between the RBS and the start codon has a strong influence on how actively a particular ORF is
translated: high complementarity and proper spacing promote active translation, whereas limited
complementarity and/or poor spacing generally support lower levels of translation.

Fig.2. Structure of messenger RNA. A polycistronic prokaryotic message with three ORFs. Each
ribosomebinding site is indicated by a purple box labeled RBS. (Source: Molecular Biology of Genes)

INITIATION OF TRANSLATION
For translation to be successfully initiated, three events must occur: the ribosome must be recruited
to the mRNA; a charged tRNA must be placed into the P-site of the ribosome; and the ribosome

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must be precisely positioned over the start codon. The correct positioning of the ribosome over the
start codon is critical because this establishes the reading frame for the translation of the mRNA.
Even a 1-base shift in the location of the ribosome would result in the synthesis of a completely
unrelated polypeptide.

Prokaryotic mRNAs are initially recruited to the small subunit by base pairing to rRNA

The assembly of the ribosome on an mRNA occurs one subunit at a time. The small subunit
associates with the mRNA first. In prokaryotes, the association of the small subunit with the mRNA is
mediated by base-pairing interactions between the RBS and the 16S rRNA (Fig.3). For ideally
positioned RBSs, the small subunit is positioned on the mRNA such that the start codon will be in
the P-site when the large subunit joins the complex. The large subunit joins its partner only at the
very end of the initiation process, just before the formation of the first peptide bond. Thus, many of
the key events of translation initiation occur in the absence of the full ribosome.

Fig. 3 The 16S rRNA interacts with the RBS to position the AUG in the P-site. (Source: Molecular
Biology of Genes)

Translation initiation is the only time a tRNA binds to the P-site without previously
occupying the A-site. This event requires a special tRNA known as the initiator tRNA, which
base-pairs with the start codon—usually AUG. Although the initiator tRNA is first charged
with a methionine, a formyl group is rapidly added to the methionine amino group by a
separate enzyme (Met-tRNA transformylase). Thus rather than methionine, the initiator
tRNA is coupled to N-formyl methionine. The charged initiator tRNA is referred to as fMet-
tRNAi f Met.
Three initiation factors direct the assembly of an Initiation Complex

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The initiation of prokaryotic translation commences with the small subunit and is catalyzed by three
translation initiation factors called IF1, IF2, and IF3. Each factor facilitates a key step in the initiation
process.
 IF1 prevents tRNAs from binding to the portion of the small subunit that will become part of
the A-site
 IF2 is a GTPase (a protein that binds and hydrolyzes GTP) that interacts with three key
components of the initiation machinery: the small subunit, IF1, and the charged initiator
tRNA (fMet-tRNAi f Met). By interacting with these components, IF2 facilitates the association
of fMet-tRNAi f Met with the small subunit and prevents other charged tRNAs from associating
with the small subunit.
 IF3 binds to the small subunit and blocks it from reassociating with a large subunit. Because
initiation requires a free small subunit, the binding of IF3 is critical for a new cycle of
translation. IF3 becomes associated with the small subunit at the end of a previous round of
translation when it helps to dissociate the 70S ribosome into its large and small subunits.
The ribosome has three sites for tRNA: the A (acceptor) site, the P (peptide) site and the E (exit)
site. Each of the initiation factors binds at, or near, one of the three tRNA binding sites on the small
subunit. Consistent with its role in blocking the binding of charged tRNAs to the A-site, IF1 binds
directly to the portion of the small subunit that will become the A-site. IF2 binds to IF1 and reaches
over the A-site into the P-site to contact the fMet-tRNAi f Met. Finally, IF3 occupies the part of the
small subunit that will become the E-site. Thus, of the three potential tRNA-binding sites on the
small subunit, only the P-site is capable of binding a tRNA in the presence of the initiation factors.
With all three initiation factors bound, the small subunit is prepared to bind to the mRNA and the
initiator tRNA. These two RNAs can bind in either order and independently of each other. Binding to
the mRNA involves base pairing between the RBS and the 16S rRNA in the small subunit.
Meanwhile, binding fMet-tRNAi f Met to the small subunit is facilitated by its interactions with IF2
bound to GTP and base pairing between the anticodon and the start codon of the mRNA. Similarly,
base pairing between the fMet-tRNAi f Met and the mRNA serves to position the start codon in the P-
site.
The last step of initiation involves the association of the large subunit to create the 70S initiation
complex (Fig. 4). When the start codon and fMet-tRNAi f Met base-pair, the small subunit undergoes
a change in conformation. This altered conformation results in the release of IF3. In the absence of
IF3, the large subunit is free to bind to the small subunit with its cargo of IF1, IF2, mRNA, and fMet-
tRNAi fMet. In particular, IF2 acts as an initial docking site of the large subunit, and this interaction
subsequently stimulates the GTPase activity of IF2-GTP. IF2 bound to GDP has reduced affinity for
the ribosome and the initiator tRNA, leading to the release of IF2-GDP as well as IF1 from the
ribosome. Thus, the net result of initiation is the formation of an intact (70S) ribosome assembled at
the start site of the mRNA with fMet-tRNAi f Met in the P-site and an empty A-site. The ribosome–
mRNA complex is now poised to accept a charged tRNA into the A-site and commence polypeptide
synthesis.

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Fig.4. A summary of translation initiation in prokaryotes. (Source: Molecular Biology of Genes)

TRANSLATION ELONGATION
Once the ribosome is assembled with the charged initiator tRNA in the P-site, polypeptide synthesis
can begin. There are three key events that must occur for the correct addition of each amino acid.
First, the correct aminoacyl-tRNA is loaded into the A-site of the ribosome as dictated by the A-site
codon. Second, a peptide bond is formed between the aminoacyl-tRNA in the A-site and the
peptide chain that is attached to the peptidyl-tRNA in the P-site. This peptidyl transferase reaction,
results in the transfer of the growing polypeptide from the tRNA in the P-site to the amino acid
moiety of the charged tRNA in the A-site. Third, the resulting peptidyl-tRNA in the A-site and its
associated codon must be translocated to the P-site so that the ribosome is poised for another cycle

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of codon recognition and peptide bond formation (Fig. 5). As with the original positioning of the
mRNA, this shift must occur precisely to maintain the correct reading frame of the message. Two
auxiliary proteins known as elongation factors control these events. Both of these factors use the
energy of GTP binding and hydrolysis to enhance the rate and accuracy of ribosome function.

Fig.5. Overview of the Elongation Cycle on the Ribosome. (Source: Molecular Biology by D.
Clark).

TERMINATION OF TRANSLATION
The ribosome’s cycle of aminoacyl-tRNA binding, peptide-bond formation, and translocation
continues until one of the three stop codons enters the A-site. It was initially postulated that there
would be one or more chain terminating tRNAs that would recognize these codons. However, this is
not the case. Instead, stop codons are recognized by proteins called release factors (RFs) that
activate the hydrolysis of the polypeptide from the peptidyl-tRNA.
There are two classes of release factors. Class I release factors recognize the stop codons and
trigger hydrolysis of the peptide chain from the tRNA in the P-site. Prokaryotes have two class I
release factors called RF1 and RF2. RF1 recognizes the stop codon UAG, and RF2 recognizes the stop
codon UGA. The third stop codon, UAA, is recognized by both RF1 and RF2.
The completed polypeptide chain is now released from the last tRNA. This is actually done by the
peptidyl transferase. Binding of the release factor activates the peptidyl transferase which
hydrolyzes the bond between the finished polypeptide chain and the tRNA in the P-site. The
polypeptide chain, the tRNA and the mRNA now leave the ribosome, which dissociates into
separate subunits. Two further factors aid in dissociation: RF3 releases RF1 or RF2 from the
ribosome and ribosome recycling factor (RRF) dissociates the large and small subunits.

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Polyribosomes:
Several ribosomes will move along the same mRNA about a hundred bases apart (Fig. 6). An mRNA
with several attached ribosomes is called a polysome (polyribosome).
Completed
Growing polypeptide
Incoming polypeptides
ribosomal
subunitsStart of End of
mRNA mRNA
An mRNA (5 end)
molecule is generally translated
(3 end) simultaneously
(a)
by several ribosomes in clusters called polyribosomes.

Ribosomes
mRNA

0.1 µm
This micrograph shows a large polyribosome in a prokaryotic
cell (TEM).
Fig. 6. A single mRNA molecule is associated with several ribosomes.

Animations:
1. https://www.youtube.com/watch?v=KZBljAM6B1s&t=2s

Section 5: Additional resources (further reading)

1. Molecular Biology of the Gene, 7th edition (2013), J. D. Watson, T. A. Baker, S. P. Bell, A.
Gann, M. Levine. and R. Losick; Cold Spring Harbor Laboratory Press. CA, USA, ISBN:
9780805395921.
2. Molecular Cell Biology, 7th edition (2012), H. Lodish, A. Berk, C. A. Kaiser, M. Krieger, M.P.
Scott, A. Bretscher, H. Ploegh and P. Matsudaira; W H Freeman & Co, ISBN: 9781429234139.
3. Molecular Biology, 2nd edition (2012), D. Clark and N. J. Pazdernik; Elsevier Academic Press.
USA, ISBN: 9780123785947.