LECT-10

mRNA processing, genetic

code, and protein synthesis
Learning Outcome (LO)
After students learn this lecture, students
are able to explain:

11.1 mRNA processing (LO 11.1)

11.2 genetic code (LO 11.2)
11.3 aspects of protein synthesis (LO 11.3)
11.4 protein synthesis (LO 11.4)
 PROCESSING OF mRNA
 There is essentially no processing of prokaryotic mRNA; it
can start to be translated before it has finished being
transcribed.
 In eukaryotes, mRNA is synthesized by RNA Pol II as
longer precursors (pre-mRNA), the population of different
pre-mRNAs being called heterogeneous nuclear RNA
(hnRNA).
 Specific proteins bind to hnRNA to form hnRNP and then
small nuclear RNP (snRNP) particles interact with hnRNP to
carry out some of the RNA processing events.
 Processing of eukaryotic hnRNA involves four events: 5-
capping, 3-cleavage and polyadenylation, splicing and
methylation.

LO 11.1: Students are able to explain mRNA processing

 PROCESSING OF mRNA
hnRNP
 RNA Pol II transcripts (hnRNA) complex with the three most
abundant hnRNP proteins, the A, B and C proteins, to form
hnRNP particles.
 These contain three copies of three tetramers and around
600–700 nucleotides of hnRNA.
 They assist RNA processing events.

LO 11.1: Students are able to explain mRNA processing

 PROCESSING OF mRNA
snRNP particles
 There are many uracil-rich snRNA molecules made by RNA
Pol II which complex with specific proteins to form snRNPs.
 The most abundant are involved in splicing, and a large
number define methylation sites in pre-rRNA.
 Those containing the sequence 5-RA(U)nGR-3’ bind eight
common proteins in the cytoplasm, become
hypermethylated and are imported back into the nucleus.

LO 11.1: Students are able to explain mRNA processing

IUPAC nucleotide IUPAC amino acid
Three letter code Amino acid
Base code
code
A Ala Alanine
A Adenine C Cys Cysteine
C Cytosine D Asp Aspartic Acid
E Glu Glutamic Acid
G Guanine
F Phe Phenylalanine
T (or U) Thymine (or Uracil) G Gly Glycine
H His Histidine
R A or G
I Ile Isoleucine
Y C or T K Lys Lysine
S G or C L Leu Leucine
M Met Methionine
W A or T
N Asn Asparagine
K G or T P Pro Proline

M A or C Q Gln Glutamine
R Arg Arginine
B C or G or T
S Ser Serine
D A or G or T T Thr Threonine

H A or C or T V Val Valine
W Trp Tryptophan
V A or C or G
Y Tyr Tyrosine
N any base
- gap
 PROCESSING OF mRNA
5’ Capping
 This is the addition of a 7-
methylguanosine nucleotide
(m7G) to the 5’-end of an
RNA Pol II transcript when it
is about 25 nt long.
 The m7G, or cap, is added
in reverse polarity (5’ to 5’),
thus acting as a barrier to 5’-
exonuclease attack, but it
also promotes splicing,
transport and translation.

LO 11.1: Students are able to explain mRNA processing

 PROCESSING OF mRNA
3’ Cleavage and polyadenylation
 Most eukaryotic pre-mRNAs are cleaved at a polyadenylation site and
poly(A) polymerase (PAP) then adds a poly(A) tail of around 250 nt to
generate the mature 3’-end.

LO 11.1: Students are able to explain mRNA processing

 PROCESSING OF mRNA
Splicing
 In eukaryotic pre-mRNA processing, intervening sequences
(introns) that interrupt the coding regions (exons) are
removed (spliced out), and the two flanking exons are
joined.
 This splicing reaction occurs in the nucleus and requires the
intron to have a 5’-GU, an AG-3’ and a branchpoint
sequence.
 In a two-step reaction, the intron is removed as a tailed
circular molecule, or lariat, and is degraded.
 Splicing involves the binding of snRNPs to the conserved
sequences to form a spliceosome in which the cleavage
and ligation reactions take place.

LO 11.1: Students are able to explain mRNA processing

 PROCESSING OF mRNA
Splicing

LO 11.1: Students are able to explain mRNA processing

 THE GENETIC CODE
Nature
 The genetic code is the way in which the nucleotide
sequence in nucleic acids specifies the amino acid
sequence in proteins.
 It is a triplet code, where the codons (groups of three
nucleotides) are adjacent (nonoverlapping) and are not
separated by punctuation (comma-less).
 Because many of the 64 codons specify the same amino
acid, the genetic code is degenerate (has redundancy).

LO 11.2: Students are able to explain the genetic code

 THE GENETIC CODE
Deciphering
 The standard genetic code was deciphered by adding homopolymers,
copolymers or synthetic nucleotide triplets to cell extracts which were
capable of limited translation.
 It was found that 61 codons specify the 20 amino acids and there are
three stop codons.

LO 11.2: Students are able to explain the genetic code

 THE GENETIC CODE
Features
 Eighteen of the 20 amino acids are specified by multiple (or
synonymous) codons which are grouped together in the
genetic code table.
 Usually they differ only in the third codon position.
 If this is a pyrimidine, then the codons always specify the
same amino acid.
 If a purine, then this is usually also true.

LO 11.2: Students are able to explain the genetic code

 THE GENETIC CODE
Effect of mutation
 The grouping of synonymous codons means that the effects
of mutations are minimized.
 Transitions in the third position often have no effect, as do
transversions more than half the time.
 Mutations in the first and second position often result in a
chemically similar type of amino acid being used.

LO 11.2: Students are able to explain the genetic code

 THE GENETIC CODE
Universality
 Until recently, the standard genetic code was considered
universal: however, some deviations are now known to
occur in mitochondria and some unicellular organisms.

LO 11.2: Students are able to explain the genetic code

 THE GENETIC CODE
ORFs
 Open reading frames are suspected coding regions usually
identified by computer in newly sequenced DNA.
 They are continuous groups of adjacent codons following a
start codon and ending at a stop codon.

LO 11.2: Students are able to explain the genetic code

 THE GENETIC CODE
Overlapping genes

 These occur when the coding region of one gene partially or

completely overlaps that of another.
 Thus one reading frame encodes one protein, and one of
the other possible frames encodes part or all of a second
protein.
 Some small viral genomes use this strategy to increase the
coding capacity of their genomes.

LO 11.2: Students are able to explain the genetic code

 ASPECTS OF PROTEIN SYNTHESIS
Codon–anticodon interaction
In the cleft of the
ribosome, an antiparallel
formation of three base
pairs occurs between the
codon on the mRNA and
the anticodon on the
tRNA.
If the 5 anticodon base is
modified, the tRNA can
usually interact with more
than one codon.

LO 11.3: Students are able to explain aspects of protein synthesis

 ASPECTS OF PROTEIN SYNTHESIS
Wobble

The wobble hypothesis describes the nonstandard base

pairs that can form between modified 5’-anticodon bases
and 3’-codon bases.
When the wobble nucleoside is inosine, the tRNA can base-
pair with three codons – those ending in A, C or U.

LO 11.3: Students are able to explain aspects of protein synthesis

 ASPECTS OF PROTEIN SYNTHESIS
Ribosome binding site

The ribosome binding site is a sequence just upstream of

the initiation codon in prokaryotic mRNA which base-pairs
with a complementary sequence near the 3’-end of the 16S
rRNA to position the ribosome for initiation of protein
synthesis.
It is also known as the Shine–Dalgarno sequence after its
discoverers.

LO 11.3: Students are able to explain aspects of protein synthesis

 ASPECTS OF PROTEIN SYNTHESIS
Polysomes

Polyribosomes (polysomes) form on an mRNA when

successive ribosomes attach, begin translating and move
along the mRNA.
A polysome is a complex of multiple ribosomes in various
stages of translation on one mRNA molecule.

LO 11.3: Students are able to explain aspects of protein synthesis

 ASPECTS OF PROTEIN SYNTHESIS
Initiator RNA

A special tRNA (initiator tRNA), recognizing the AUG start

codon, is used to initiate protein synthesis in both
prokaryotes and eukaryotes.
In prokaryotes, the initiator tRNA is first charged with
methionine by methionyl-tRNA synthetase.
The methionine residue is then converted to N-
formylmethionine by transformylase.
In eukaryotes, the methionine on the initiator tRNA is not
modified.
There are structural differences between the E. coli initiator
tRNA and the tRNA that inserts internal Met residues.

LO 11.3: Students are able to explain aspects of protein synthesis

 ASPECTS OF PROTEIN SYNTHESIS
Initiator RNA

LO 11.3: Students are able to explain aspects of protein synthesis

 MECHANISM OF PROTEIN SYNTHESIS
Overview

There are three stages of protein synthesis:

 initiation – the assembly of a ribosome on an mRNA;
 elongation – repeated cycles of amino acid delivery,
peptide bond formation and movement along the mRNA
(translocation);
 termination – the release of the polypeptide chain.

LO 11.4: Students are able to explain mechanism of protein synthesis

 MECHANISM OF PROTEIN SYNTHESIS
Initiation
In prokaryotes, initiation requires the large and small
ribosome subunits, the mRNA, the initiator tRNA, three
initiation factors (IFs) and GTP.
IF1 and IF3 bind to the 30S subunit and prevent the large
subunit binding. IF2 + GTP can then bind and will help the
initiator tRNA to bind later.
This small subunit complex can now attach to an mRNA via
its ribosome-binding site.
The initiator tRNA can then base-pair with the AUG initiation
codon which releases IF3, thus creating the 30S initiation
complex.
The large subunit then binds, displacing IF1 and IF2 + GDP,
giving the 70S initiation complex which is the fully
assembled ribosome at the correct position on the mRNA.
LO 11.4: Students are able to explain mechanism of protein synthesis
 MECHANISM OF PROTEIN SYNTHESIS
Initiation

LO 11.4: Students are able to explain mechanism of protein synthesis

 MECHANISM OF PROTEIN SYNTHESIS
Elongation
Elongation involves the three factors (EFs), EF-Tu, EF-Ts
and EF-G, GTP, charged tRNAs and the 70S initiation
complex (or its equivalent).
It takes place in three steps.
 A charged tRNA is delivered as a complex with EF-Tu
and GTP. The GTP is hydrolyzed and EF-Tu:GDP is
released which can be re-used with the help of EF-Ts
and GTP (via the EF-Tu–EF-Ts exchange cycle).
 Peptidyl transferase makes a peptide bond by joining
the two adjacent amino acids without the input of more
energy.
 Translocase (EF-G), with energy from GTP, moves the
ribosome one codon along the mRNA, ejecting the
uncharged tRNA and transferring the growing peptide
chain to the P-site.
LO 11.4: Students are able to explain mechanism of protein synthesis
 MECHANISM OF PROTEIN SYNTHESIS
Elongation

LO 11.4: Students are able to explain mechanism of protein synthesis

 MECHANISM OF PROTEIN SYNTHESIS
Termination
Release factors (RF1 or RF2)
recognize the stop codons and,
helped by RF3, make peptidyl
transferase join the polypeptide
chain to a water molecule, thus
releasing it.
Ribosome release factor helps
to dissociate the ribosome
subunits from the mRNA.

LO 11.4: Students are able to explain mechanism of protein synthesis

 LECT-11
Gene manipulation