Translation

In this lesson, you will learn how proteins are made from the code carried by the messenger RNA. In the first
part, you will be introduced to the structures and functions of the essential components of protein synthesis
like the mRNA, tRNA, aminoacyl-tRNA synthetase, and the ribosome. Then in the second part, the molecular
details of the overall process of translation will be elaborated.

Jayzon G. Bitacura, MSc., RMicro

Assistant Professor III
Department of Biological Sciences
College of Arts and Sciences
Visayas State University
 Learning Outcomes
At the end of this lesson, you will be able to:
1. identify the molecular machinery that governs protein synthesis.
2. describe the structures and functions of each of these machineries; and
3. discuss the overall process of translation.

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Motivation
Provide the polypeptide chain encoded by the given mRNA by referring to the genetic code provided.
Use the three-letter symbol for each amino acid.

5’ A U G C C G A A U G G U C U A C G C G C A U A A 3’

___________________________________________________

You are correct! The polypeptide chain encoded by the given

mRNA is

Met – Pro – Asn – Gly – Leu – Arg – Ala

How is the cell able to use the code carried by

the mRNA to synthesize a polypeptide chain?

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Translation
● Translation is a process wherein the information contained within the
order of nucleotides in messenger RNA (mRNA) transcribed from the
genes is interpreted to produce the linear sequences of amino acids in
polypeptides/proteins.
● Scientists thought that it would seem unlikely that direct interactions
between the mRNA template and the amino acids could be responsible
for the specific and accurate ordering of amino acids in a polypeptide.
● Thus, in 1955 Francis H. Crick proposed that before their incorporation
into polypeptides, amino acids must attach to a special adaptor
molecule that is capable of directly interacting with and recognizing the
three-nucleotide-long coding units of the mRNA.
● Crick imagined that the adaptor would be an RNA molecule because it
would need to recognize the code by Watson-Crick base-pairing rules.
● It was shown by Paul C. Zamecnik and Mahlon B. Hoagland that amino
acids are attached to a class of RNA molecules before they are
incorporated into proteins.
○ These types of RNAs are called transfer RNAs (tRNAs).
○ They are named as such because the amino acids attached to
them are subsequently transferred to the growing polypeptide
chain.

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 Translation Machinery
● Four primary components compose the machinery responsible
for translating the language of mRNAs into the language of
proteins.
○ These components are mRNAs, tRNAs,
aminoacyl-tRNA synthetases, and the ribosomes.
● Together, these components translate a code written in a
four-base alphabet into a second code that uses the 20 amino
acids to write the language.
● The mRNA carries the information to be interpreted by the
translation machinery and serves as the template for the
translation process.
○ The mRNA region that codes for the protein consists of
an ordered series of three-nucleotide units called
codons that specify the sequence of amino acids.
● The tRNAs provide the physical link between the amino acids
being added to the growing polypeptide chain and the codons
in the mRNA.
● Enzymes called aminoacyl-tRNA synthetases couple amino
acids to specific tRNAs that recognize the appropriate
codon/s.
● The final major player in translation is the ribosome.
○ It coordinates the correct recognition of the mRNA by
each tRNA and catalyzes peptide-bond formation
between the growing polypeptide chain and the amino
acid attached to the selected tRNA.
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 The messenger RNA (mRNA)
● The protein-coding region of each mRNA is composed of a
continuous, non-overlapping chain of codons called the open
reading frame (ORF), which specifies a protein.
● Each ORF starts and ends at internal sites embedded within the
mRNA.
● This means that the ends of an ORF are distinct from the ends
of the mRNA.
● Translation starts at one end (the 5’ end) of the ORF and
continues one codon at a time to the other end, which is the 3’
end.
● The first and last codons of an ORF are known as the start and
stop codons.
● Eukaryotes always use 5’-AUG-3’ as their start codon.
● Bacteria also usually use this, but they could also use 5’-GUC-3’
and sometimes even 5’-UUG-3’.
● The start codon functions for specifying the first amino acid to
be incorporated into the growing polypeptide chain, and it also
defines the reading frames for all subsequent codons.
● On the other hand, the stop codons 5’-UAG-3’, 5’-UGA-3’, and
5’-UUA-3’ define the end of the ORF and signal the termination
of the polypeptide synthesis.

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mRNA
● In all organisms, mRNAs contain at least one
ORF.
● The ORFs’ number per mRNA is different
between prokaryotes and eukaryotes.
● Eukaryotic mRNAs almost always contain a
single ORF.
○ This is referred to as monocistronic
mRNAs.
● In contrast, prokaryotic mRNAs frequently
contain two or more ORFs and hence can
encode multiple polypeptide chains.
○ These types of mRNAs are known as
polycistronic mRNAs.

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 mRNA
● Recruitment of the ribosomes to the mRNA is necessary so that translation can occur.
● To facilitate binding by a ribosome, many prokaryotic ORFs contain a ribosome binding site (RBS), which is a short
sequence upstream of the start codon and is also referred to as Shine-Dalgarno sequence.
● 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 rRNA.
● The RBS base pairs with this RNA, thereby aligning the ribosome with the beginning of the ORF.
● The core of this region of the 16S rRNA has the sequence 5’-CCUCCU-3’ while prokaryotic RBS is most often a
subset of the sequence 5’-AGGAGG-3’.

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mRNA ● Unlike that of prokaryotes, eukaryotic
mRNAs recruit ribosomes through a specific
post-transcriptional chemical modification
called the 5’ cap, which is located at the
extreme 5’ end of the mRNA (please recall
the previous lesson).
● Once the ribosome is bound to the mRNA, it
moves in a 5’ -to- 3’ direction until it
encounters a 5’-AUG-3’ start codon.
○ This process is called scanning.

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The transfer RNA (tRNA)
● The center of protein synthesis is the “translation” of
nucleotide sequence information into amino acids.
● This process is done by tRNA molecules, which act as
adaptors between the amino acids they specify and the
codons.
● There are several types of tRNA molecules.
● Each of these types is attached to a specific amino acid,
and each of them recognizes a particular codon, or
codons, in the mRNA.
● All tRNAs have certain features in common.
○ First, all tRNAs end at the 3’ terminus with the
sequence 5’-CCA-3’ which is attached to the
cognate amino acid.
○ Second is the presence of several unusual bases
in their primary structure which are created
posttranscriptionally by enzymatic modification of
normal bases in the polynucleotide chain.
■ Examples are pseudouridine (YU) and
dihydrouridine (D), which are derived from
uridine.
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tRNA
● tRNA molecules show a
characteristic and highly conserved
pattern of single-stranded and
double-stranded regions (due to
self-complementarity) that can be
illustrated as a cloverleaf.
● The principal features of the tRNA
cloverleaf are an acceptor stem,
three stem-loops (referred to as YU
loop, the D loop, and the anticodon
loop), and a variable loop.

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tRNA
● The acceptor stem is the attachment site for the cognate amino
acid.
○ It is formed by pairing between the 5'- and 3'-ends of the
tRNA molecule.
○ The 5'-CCA-3' end protrudes from this double-stranded stem
and is preceded by the unpaired discriminator base.
● The ѰU loop is the most 3' proximal loop and contains
pseudouridine frequently found within the sequence 5'-TYUCG-3'.
● The D loop is the most 5'-proximal loop and contains dihydrouridine
residues.
● The anticodon loop contains the anticodon sequence that is
complementary to the codon in the mRNA.
○ Codon and anticodon triplets interact by RNA-RNA base
pairing.
○ Because codons in the mRNA are read from 5' -to- 3',
anticodons are oriented and written in the 3' -to- 5' direction.
● The variable loop is located between the anticodon loop and the YU
loop.
○ It varies in size from 3 to 21 nucleotides.
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● Cells do not have a specific tRNA for every codon as this would require 61

●
tRNAs.
Instead, it was discovered that certain tRNA species of known sequence tRNA
can recognize several different codons.
● Thus, the genetic code is described as degenerate.
● Furthermore, some tRNAs do not have one of the four classical bases but Base in Base in
have inosine instead in the first position of the anticodon.
Anticodon Codon
● Like all the other minor tRNA bases (modified bases), inosine arises
through enzymatic modification of a base in a completed tRNA chain (it is
not directly incorporated during transcription of the tRNA gene).
G U or C
● The wobble theory (developed by F. Crick in 1966) states that the base at
the 5'-end of the anticodon is not as spatially confined as the other two, A U
allowing it to form hydrogen bonds with any of several bases located at the
3'-end of the codon.
● This is because sliding can occur and that alternative hydrogen bond
C G
formation is possible.
● However, not all combinations are possible: U A or G
● The pairings that are allowed by the wobble theory are those that produce
ribose-ribose distances close to that of the standard A:U, or G:C bp.
I A, U or C
● Purine-purine (except for A:I pairs) or pyrimidine-pyrimidine pairs would give
rise to ribose-ribose distances that are too short or too long, respectively.
● The "wobble movement" is restricted to the first position of the anticodon
because the other two positions are more "stacked" and surrounded by
modified, bulky purine residues that further restrict their movement.
● The wobble theory rules do not permit any single tRNA molecule to
recognize four different codons.
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The Aminoacyl-tRNA Synthetase
● Charged tRNA molecules are those
tRNAs with an already attached amino
acid.
● The requirement for charging are the
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.
● The enzyme called aminoacyl-tRNA
synthetase is responsible for this
catalysis.

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Charging tRNAs ● Two enzymatic steps are performed by all
aminoacyl-tRNA synthetases during the attachment of
an amino acid to tRNA.
● Step one is adenylylation in which the amino acid reacts
with ATP to become adenylylated with the concomitant
release of pyrophosphate.
● Adenylylation refers to the transfer of AMP, as opposed
to adenylation which is the transfer of adenine.
● 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.
● Each of the 20 amino acids is attached to the
appropriate tRNA by a single, dedicated tRNA
synthetase.
● It is not uncommon for one synthetase to recognize and
charge more than one tRNA (known as isoaccepting
tRNAs) because most amino acids are specified by more
than one codon.

How does the aminoacyl-tRNA synthetase recognize the correct amino acid to charge in a
particular tRNA?
● Studies have indicated that the determinants for the specificity of tRNA recognition
by aminoacyl-tRNA synthetase are found at two distant sites on the molecule.
● These are the acceptor stem and the anticodon loop.
● The aminoacyl-tRNA synthetase must couple the proper amino acid with its
cognate tRNA because the ribosome cannot distinguish between correctly and
incorrectly charged tRNAs.
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The Ribosome
● Directing the synthesis of proteins is the
macromolecular machine called the
ribosome.
● It is larger and more complex than the
minimal machinery required for DNA and
RNA synthesis.
● In prokaryotes, the ribosome can commence
translation of the mRNA as it emerges from
the RNA polymerase.
● This is because the transcription machinery
and the translation machinery are located in
the same compartment.
● In eukaryotes, on the other hand, the two
processes occur at different locations in the
cell and thus are completely separate from
each other.
● Eukaryotic transcription occurs in the
nucleus, while translation occurs in the
cytoplasm.

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Ribosome
● The ribosome is composed of an assembly of RNA and protein known as the large and small subunits.
● The large subunit contains the peptidyl transferase center, which is responsible for the formation of
peptide bonds.
● The small subunit contains the decoding center in which charged tRNAs read or decode the codon units
of the mRNA.
● By convention, the large and small subunits are named according to the velocity of their sedimentation
when subjected to centrifugal force.
● The unit of measure sedimentation velocity is the Svedberg.
● Prokaryote vs Eukaryote?

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Ribosome
How is the peptide bond formed in a growing polypeptide chain?
● The ribosome also catalyzes the formation of a peptide
bond which is called peptidyl transferase reaction.
● This enzymatic reaction occurs between the amino acid
residue at the carboxy-terminal end of the growing
polypeptide and the incoming amino acid to be added to
the chain.
● The growing chain attached to tRNA is called the
peptidyl-tRNA while a tRNA with attached amino acid is
called aminoacyl-tRNA.
● The ribosome must be able to bind to at least two tRNAs
simultaneously to perform the peptidyl transferase
reaction.
● The ribosome contains three tRNA-binding sites, called
the A-, P-, and E sites.
● The A-site is the site for the binding of the
aminoacyl-tRNA, the P-site is the binding site for the
peptidyl-tRNA, and the E-site is the site for binding the
tRNA that is released after the growing polypeptide chain
has been transferred to the aminoacyl-tRNA.

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The Process of Translation

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 Summary Questions

● What is translation?
● What are the important translation
machineries? What are the
molecular mechanisms of their
function?
● What are the stages of translation?
Discuss the molecular process.

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Contact information:
jayzon.bitacura@vsu.edu.ph

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