MOLECULAR BIOLOGY- Unit IV

RNA metabolism class I

 Outline

➢ Overview and interconnection of 4 molecular genetic processes

➢ Prokaryotic and eukaryotic transcription

➢ Basic terminology and conventions used in RNA metabolism

➢ Enzymology, types of RNA polymerases involved

➢ Initiation, elongation and termination of transcription

➢ Post transcriptional modifications

Overview of four basic molecular genetic processes
During transcription of a protein-coding
gene by RNA polymerase, the four-base
DNA code specifying the amino acid
sequence of a protein is copied, or
transcribed, into a precursor messenger
RNA (pre-mRNA) by the polymerization
of ribonucleoside triphosphate
monomers (rNTPs).
Removal of noncoding sequences and
other modifications to the pre-mRNA 2 ,
collectively known as RNA processing,
produce a functional mRNA, which is
transported to the cytoplasm.
During translation, the four-base code
of the mRNA is decoded into the 20–
amino acid language of proteins
During DNA replication 4, which occurs
only in cells preparing to divide,
deoxyribonucleoside triphosphate
monomers (dNTPs) are polymerized to
yield two identical copies of each
chromosomal DNA molecule. Each
daughter cell receives one of the
identical copies.
• Termination relies on
terminator sequence
• Termination relies on no
terminator sequence
A Template DNA Strand Is Transcribed into a Complementary
RNA Chain by RNA Polymerase
One DNA strand acts as a template, determines order of ribo NTPs (rNTP) monomers in the
complementary RNA chain.

Template DNA strand base-pair with complementary incoming rNTPs, which are then joined in a
polymerization reaction catalyzed by RNA polymerase.
The polymerization reaction similar to DNA pol
involves a nucleophilic attack by the 3′ oxygen in the
growing RNA chain on the α phosphate of the next
nucleotide precursor to be added, which results in
the formation of a phosphodiester bond and the
release of pyrophosphate (PPi).

As a consequence of this mechanism, RNA

molecules are always synthesized in the 5′→3′
direction; so by convention, RNA represented 5′→3′
 Conventions for describing RNA transcription

Top: The DNA nucleotide where RNA polymerase begins transcription is designated +1.
The direction the polymerase travels on the DNA is “downstream,” and downstream
bases are marked with positive numbers.
The opposite direction is “upstream,” and upstream bases are marked with negative
numbers.
Some important gene features lie upstream of the transcription start site, including the
promoter sequence that localizes RNA polymerase to the gene.

Bottom: The DNA strand that is being transcribed is the template strand; its complement
is the nontemplate strand. The RNA being synthesized is complementary to the template
strand and is therefore identical with the nontemplate strand sequence, except with
uracil in place of thymine.
Q. The antisense strand of a segment of DNA has
the following sequence.
3’-AGGTATCCCATC-5’.
What will be the corresponding sequence of the
mRNA transcribed from this segment of DNA?

A 5’-AGGUAUCCCAUC-3’
B 3’-UGGTUTCGCATT-5’
C 3’-ACCUAUGCGAUC-5’
D 5’-UCCAUAGGGUAG -3’

5’-UCCAUAGGGUAG -3’
Q. A sense strand of DNA has a sequence:
3’ – CAGCGACTCGGGGTA – 5’. What is the RNA
sequence for this strand?

3’ – CAGCGACUCGGGGUA – 5’

Correct: 5’ – AUGGGGCUCAGCGAC – 3’
The Transcription Reaction Has Three Stages

Transcription has three stages:

1. The enzyme binds to the promoter

and melts DNA and remains stationary
during initiation;

2. moves along the template during

elongation;

3.and dissociates at termination.

Bacterial RNA Polymerase Consists of Multiple Subunits

, , ', and  have rather constant

sizes in different bacterial species,
but  varies more widely.

The several subunits of the

bacterial RNA polymerase
give the enzyme the shape of
a crab claw. The pincers are
formed from the large  and ’
subunits. The subunits are
shown in the same colors in
the schematic and the ribbon
structure.
 RNA Synthesis Begins at Promoters

Sequences of the non-template strand (5’ → 3’) are shown (5’ → 3’ direction is the convention for
representations of this kind).
The sequences differ from one promoter to the next, but comparisons of many promoters reveal
similarities, particularly in the -10 and -35 regions.

The sequence element UP (-40 to -60), not present in all E. coli promoters, is shown in the P1
promoter for the highly expressed rRNA gene rrnB. UP elements, strongly stimulate transcription at
the promoters that contain them. The UP element in the rrnB P1 promoter encompasses the region
between -38 and -59.

The consensus sequence for E. coli promoters recognized by 70 is shown second from the top.
Spacer regions contain slightly variable numbers of nucleotides (N). Only the first nucleotide
coding the RNA transcript (at position 1) is shown
 Footprint analysis

Footprint analysis of the RNA polymerase–binding site on a DNA fragment. Separate

experiments are carried out in the presence (+) and absence (-) of the polymerase.
Identifying lac promoter

Footprinting results of RNA polymerase binding to the

lac promoter.

In this experiment, the 5’ end of the nontemplate

strand was radioactively labeled.

Lane C is a control in which the labeled DNA

fragments were cleaved with a chemical reagent that
produces a more uniform banding pattern.
The structure of sigma factor in the context of the holoenzyme: -10 and -35 interactions.

Sigma factor is extended and its domains are connected by flexible linkers.
 Eukaryotes have 3 different RNA polymerases
Form Product Location, Promoter  Amanitin

I 5.8S rRNA, 18S Nucleolus, variable from species to another Insensitive

rRNA, 28S rRNA
II Nuclear genes Nucleoplasm, TATA box (eukaryotic consensus sequence Highly sensitive
mRNA TATA(A/T)A(A/T) (A/G)) near base pair -30 and an Inr sequence (initiator)
near the RNA start site at +1)

III tRNA, 5SrRNA Nucleoplasm, located within the gene itself or upstream Less sensitive
Transcription initiation and elongation by E. coli RNA polymerase.

Initiation divided into two phases,

binding and initiation.
In the binding phase, the initial
interaction of the RNA polymerase
with the promoter leads to formation
of a closed complex, in which the
promoter DNA is stably bound but
not unwound.
A 12 to 15 bp region of DNA from
within the -10 region to position +2
or +3 is then unwound to form an
open complex.
Additional intermediates (not
shown) have been detected in the
pathways leading to the closed and
open complexes, along with several
changes in protein conformation.
The initiation phase encompasses
transcription initiation and promoter
clearance (steps 1 through 4 here).

Once elongation commences, the  subunit is released and it is replaced by the protein NusA. The
polymerase leaves the promoter and becomes committed to elongation of the RNA (step 5). When
transcription is complete, the RNA is released, the NusA protein dissociates, and the RNA polymerase
dissociates from the DNA (step 6). Another subunit binds to the RNA polymerase and the process begins
again
Elongation by RNA polymerase.
synthesis of an RNA strand
complementary to one of two DNA
strands in a double helix, the DNA is
transiently unwound.

Catalytic mechanism of RNA synthesis

by RNA polymerase. Note that this is
essentially the same mechanism used
by DNA polymerases (unit III).

Reaction involve 2 Mg+2 ions,

coordinated to the phosphate groups of
the incoming nucleoside triphosphates
(NTPs) and to three Asp residues,
which are highly conserved in the RNA
polymerases of all species.

One Mg+2 ion facilitates attack by the 3’-

hydroxyl group on the phosphate of the
NTP; the other Mg+2 ion facilitates
displacement of the pyrophosphate, and
both metal ions stabilize the Penta
covalent transition state.
(b) About 17 bp of DNA are unwound at
any given time. RNA polymerase and the
transcription bubble move from left to
right.
The DNA is unwound ahead and rewound
behind as RNA is transcribed. As the
DNA is rewound, the RNA-DNA hybrid is
displaced and the RNA strand is
extruded.
(c) Movement of an RNA polymerase
along DNA tends to create positive
supercoils (overwound DNA) ahead of
the transcription bubble and negative
supercoils (underwound DNA) behind it.
The RNA polymerase is in close contact
with the DNA ahead of the transcription
bubble as well as with the separated DNA
strands and the RNA within and
immediately behind the bubble.
A channel in the protein funnels new
NTPs to the polymerase active site. The
polymerase footprint encompasses about
35 bp of DNA during elongation.
Transcription termination (a)  independent termination. RNA
polymerase pauses at a variety of
DNA sequences, some of which
are terminators. One of two
outcomes may happen: i. the
polymerase bypasses the site and
continues on its way, or ii. the
complex undergoes a
conformational change
(isomerization). Latter results in,
intramolecular pairing of
complementary sequences in the
newly formed RNA transcript and
hairpin that disrupts the RNA-DNA
hybrid or the interactions between
RNA and polymerase, or both,
resulting in isomerization. An AU
hybrid region at the 3 end of the
new transcript is relatively unstable,
and the RNA dissociates from the
complex completely, leading to
termination. This is the usual
outcome at terminators. At other
pause sites, the complex may
escape after the isomerization step
to continue RNA synthesis.

(b) -dependent termination. RNAs that include a rut site (purple) recruit the helicase. The helicase
migrates along the mRNA in the 5 → 3 direction and separates it from the polymerase.
Eukaryotic Cells Have Three Kinds of Nuclear RNA Polymerases
Eukaryotic transcriptional machinery is much more complex than that in bacteria.

Eukaryotes have 3 RNA polymerases, designated I, II, and III; share common subunits but
perform different functions; recruitment is based on specific promotor sequence
Form Product Location, Promoter  Amanitin

I 5.8S rRNA, 18S Nucleolus, variable from species to another Insensitive

rRNA, 28S rRNA
II Nuclear genes Nucleoplasm, TATA box (eukaryotic consensus sequence Highly sensitive
mRNA TATA(A/T)A(A/T) (A/G)) near base pair -30 and an Inr sequence (initiator)
near the RNA start site at +1)

III tRNA, 5SrRNA Nucleoplasm, located within the gene itself or upstream Less sensitive

Scientist X has isolated 3 different types of RNA polymerases from

eukaryotic cells. Design an experiment to distinguish different classes
of these RNA polymerases?
Eukaryotic Cells Have Three Kinds of Nuclear RNA Polymerases

RNA Polymerase I Has a bipartite

promoter -
consists of a core promoter and
an upstream promoter element
(UPE).

The factor UBFl wraps DNA

around a protein structure to bring
the core and UPE into proximity.

SLl includes the factor TBP that is

involved in initiation by all three
RNA polymerases.

RNA polymerase I binds to the

UBF1-SL1 complex at the core
promoter.
RNA polymerase III has two types of promoters
Internal promoters have
short consensus sequences
located within the
transcription unit and cause
initiation to occur at a fixed
distance upstream.

Upstream promoters contain

three short consensus
sequences upstream of the
start point that are bound
by transcription factors.

TFIIIA and TFIIIC bind to the

consensus sequences and
enable TFIIIB to bind at the
start point.

TFIIIB has TBP as one

subunit and enables RNA
polymerase to bind.
RNA polymerase II has Inr, TATA and DPE elements
RNA polymerase II requires general
transcription factors (called TFIIX) to initiate
transcription.

RNA polymerase II promoters frequently

have a short conserved sequence
Py2CAPy5 (the initiator Inr) at the start
point.

The TATA box is a common component of

RNA polymerase II promoters and consists
of an A-T -rich octamer located ~25 bp
upstream of the start point.

The downstream promoter element (DPE)

is a common component of RNA
polymerase II promoters that do not contain
a TATA box.

A core promoter for RNA polymerase II

includes the Inr and, commonly, either a
TATA box or a DPE. It may also contain
other minor elements.
RNA polymerase II is central to eukaryotic gene expression

RBP1

A simulation of purified yeast

RNA polymerase II run on an
RBP2 SDS gel to separate the
subunits by size

Some subunits are common to

RBP3 and 11 all classes of eukaryotic RNA
polymerases and some are
related to bacterial RNA
polymerase.
Assembly of RNA Polymerase and Transcription Factors at a
Promoter

The formation of a closed complex begins Counting all the subunits of the various
when the TATA-binding protein (TBP) binds essential factors (excluding TFIIA and some
to the TATA box. subunits of TFIID), Minimal active assembly of
The sequence elements that direct the Pol II has more than 30 polypeptides.
binding of TFIID at TATA-less promoters Structural studies by Roger Kornberg and his
are poorly understood. TBP is bound in collaborators have provided a more detailed
turn by the transcription factor TFIIB, which look at the core structure of RNA polymerase
also binds to DNA on either side of TBP II during elongation.
RNA polymerase II requires an array of other proteins, called transcription factors, in
order to form the active transcription complex
 Sequential assembly of
TBP (often with TFIIA),
Transcription at RNA TFIIB, TFIIF plus Pol II,
polymerase II promoters TFIIE, and TFIIH results in
a closed complex.
Within the complex, the
DNA is unwound at the Inr
region by the helicase
activity of TFIIH and
perhaps of TFIIE, creating
an open complex.
The carboxyl-terminal
domain of the largest Pol II
subunit is phosphorylated
by TFIIH, and the
polymerase then escapes
the promoter and begins
transcription.
Elongation is accompanied
by the release of many
transcription factors and is
also enhanced by
elongation factors.
After termination, Pol II is
released,
dephosphorylated, and
recycled
An Overview of Transcription and Translation in Both
Prokaryotic and Eukaryotic Cells
(1) DNA-dependent RNA polymerases (or RNA polymerases) are responsible for transcription in
both prokaryotes and eukaryotes.

These enzymes incorporate nucleotides into a strand of RNA from a DNA template.

The promoter is where the enzyme binds prior to initiating transcription.

– The enzyme require the help of transcription factors to recognize the promoter

(2) The newly synthesized RNA chain grows in a 5’ to 3’ direction antiparallel to the DNA.
– RNA polymerase must be processive – remain attached to DNA over long stretches.
– RNA polymerase must be able to move from nucleotide to nucleotide.

Nucleotides enter the polymerization reaction as trinucleotide precursors. The reaction is driven
forward by the hydrolysis of a pyrophosphate: PP i -> 2P i

(3) Once polymerase has finished adding nucleotides, the DNA-RNA hybrid dissociates and the
DNA double helix reforms.
There are two enzymatic activities of RNA polymerase: digestion of incorrect nucleotides and
polymerization.

(4) Transcription in Bacteria – There is only one type of RNA polymerase in prokaryotes: five
subunits associated to form a core enzyme. – Transcription-competent cells also have a sigma
factor attached to the RNA polymerase before attaching to DNA.
An Overview of Transcription and Translation in Both
Prokaryotic and Eukaryotic Cells

5) Bacterial promoters are located upstream from the site of initiation. – Two conserved regions: –
35 element (consensus sequence) and Pribnow box (bacterial homologue of TATA box).
Differences in the DNA sequences at both –35 element and the Pribnow box may regulate gene
expression. Termination in bacteria can either require a rho factor protein or may reach a
terminator sequence without rho.

6) Transcription and Processing in Eukaryotic Cells – There are three types of RNA polymerases
in eukaryotes. Most rRNAs are transcribed by RNA polymerase I. mRNAs are transcribed by RNA
polymerase II. tRNAs are transcribed by RNA polymerase III.

(7) Transcription factors regulate the activity of RNA polymerases. Newly transcribed RNAs are
processed.
– A primary transcript (or pre-RNA) is the initial RNA molecule synthesized.
– A transcription unit is the DNA segment corresponding to a primary transcript.
– A variety of small RNAs are required for RNA processing.
• Termination relies on
terminator sequence
• Termination relies on no
terminator sequence
 RNA processing

Several of the enzymes that catalyze these reactions consist of RNA rather than protein.
The discovery of these catalytic RNAs, or ribozymes, has brought a revolution in thinking
about RNA function and about the origin of life

Many of the RNA molecules in bacteria and virtually all RNA molecules in eukaryotes are
processed to some degree after synthesis

Primary transcript is the newly synthesized RNA. eukaryotic mRNAs, tRNAs of both
bacteria and eukaryotes undergo extensive processing
mRNA processing

Formation of the primary transcript

and its processing during
maturation of mRNA in a eukaryotic
cell.

The 5’ cap (red) is added before

synthesis of the primary transcript
is complete.

A noncoding end sequence (intron)

following the last exon is shown in
orange.

Splicing can occur either before or

after the cleavage and
polyadenylation steps.

All the processes shown here take

place in the nucleus.
The 5’ cap of mRNA
7 6
5 1
8
4 2
9
3
(a) 7-Methylguanosine (m7 G) is
joined to the 5’ end of almost all
eukaryotic mRNAs in an unusual 5’,5’-
triphosphate linkage.

Methyl groups (light red) are often

found at the 2’ position of the first and
second nucleotides.

RNAs in yeast cells lack the 2’-methyl

groups.

The 2’-methyl group on the second

nucleotide is generally found only in
RNAs from vertebrate cells.

(b) Generation of the 5’ cap involves

four to five separate steps (adoHcy is
S-adenosylhomocysteine)
The 5’ cap of mRNA

Synthesis of the cap is carried out by enzymes

tethered to the CTD of Pol II.

The cap remains tethered to the CTD through an

association with the cap-binding complex (CBC)
 Splicing History
When Whom What
1967 Carl Woese, RNA could act as catalyst
Francis Crick and
Leslie Orgel
1970s Thomas Cech First studied excision of introns in rRNA
gene, in Tetrahymena thermophila. Found
cell extract is not needed for intron
splicing, while trying to purify the enzyme.
They could not purify any protein, finally
proposed intron is capable of doing
splicing.
1981-82 Sidney Altman Isolated enzyme Rnase P, capable of
processing tRNA. Surprisingly they
found RNAse P contained RNA in
addition to protein. Later he proved
that protein component is not
necessary for processing
1989 Cech and Awarded Nobel prize
Altman
 Ribozymes

What is a Ribozyme?

Enzyme

Not a protein

1989 Nobel Prize

In Chemistry

Sid Altman Tom Cech

 RNA Catalyzes the Splicing of Introns

There are four classes of introns. The first two, the group I and group II introns

Third is spliceosomal introns, because their removal occurs within and is catalyzed by a large
protein complex called a spliceosome. nuclear mRNA primary transcripts are processed in this way

Fourth class of introns, found in certain tRNAs, is distinguished from the group I and II introns in
that the splicing reaction requires ATP and an endonuclease. The splicing endonuclease cleaves
the phosphodiester bonds at both ends of the intron, and the two exons are joined by a mechanism
similar to the DNA ligase reaction

Transesterification reaction. Shown

here is the first step in the two-step
splicing of group I introns. In this
example, the 3’ OH of a guanosine
molecule acts as nucleophile,
attacking the phosphodiester linkage
between U and A residues at an exon-
intron junction of an mRNA molecule
Splicing mechanism of group I introns

The nucleophile in the first step may be

guanosine, GMP, GDP, or GTP. The
spliced intron is eventually degraded
Splicing mechanism of group II introns

The chemistry is similar to

that of group I intron splicing,
except for the identity of the
nucleophile in the first step
and formation of a lariat like
intermediate, in which one
branch is a 2’,5’-
phosphodiester bond.
Spliciosome mediated splicing
Splicing mechanism in mRNA primary transcripts.
(a) RNA pairing interactions in the formation of
spliceosome complexes. The U1 snRNA has a
sequence near its 5’ end that is complementary to
the splice site at the 5’ end of the intron. Base
pairing of U1 to this region of the primary
transcript helps define the 5’ splice site during
spliceosome assembly ( is pseudouridine). U2 is
paired to the intron at a position encompassing the
A residue (shaded light red) that becomes the
nucleophile during the splicing reaction. Base
pairing of U2 snRNA causes a bulge that
displaces and helps to activate the adenylate, the
2’ OH of which will form the lariat structure through
a 2’,5’-phosphodiester bond.
(b) Assembly of spliceosomes. The U1 and U2
snRNPs bind, then the remaining snRNPs (the
U4-U6 complex and U5) bind to form an inactive
spliceosome. Internal rearrangements convert this
species to an active spliceosome in which U1 and
U4 have been expelled and U6 is paired with both
the 5’ splice site and U2. This is followed by the
catalytic steps, which parallel those of the splicing
of group II introns.
(c) Coordination of splicing and transcription
brings the two splice sites together. The
spliceosome is much larger than shown in fig
 Applications

Ribozyme structure could be engineered to a therapeutic RNA

The therapeutic RNA could replace the disease specific RNA, as shown in the figure

Finally the target RNA specific transgene is expressed, nullifying disease specific effects
Q. Which of the splicing mechanism involves formation of 3
phosphodiester bonds?

a. Group 1
b. Group 2
c. Group 3
Addition of the poly(A) tail to the
primary RNA transcript of eukaryotes.
Pol II synthesizes RNA beyond the segment of
the transcript containing the cleavage signal
sequences, including the highly conserved
upstream sequence (5’)AAUAAA.

This cleavage signal sequence is bound by an

enzyme complex that includes an
endonuclease, a polyadenylate polymerase,
and several other multi subunit proteins
involved in sequence recognition, stimulation
of cleavage, and regulation of the length of the
poly(A) tail, all of which are tethered to the
CTD.
The RNA is cleaved by the endonuclease at a
point 10 to 30 nucleotides 3’ to (downstream
of) the sequence AAUAAA. The polyadenylate
polymerase synthesizes a poly(A) tail 80 to
250 nucleotides long, beginning at the
cleavage site
catalyzed the reaction:
RNA + nATP → RNA - (AMP)n + nPPi ; where n =
80 to 250. This enzyme does not require a template
but does require the cleaved mRNA as a primer.
 RNA editing and transport

Some mRNAs are edited before translation. RNA editing can involve the addition, deletion, or
alteration of nucleotides in the RNA in a manner that affects the meaning of the transcript when it is
translated. Addition or deletion of nucleotides has been most commonly observed in RNAs
originating from the mitochondrial and chloroplast genomes of eukaryotes. The reactions require a
special class of RNA molecules encoded by these same organelles, with sequences
complementary to the edited mRNAs. These guide RNAs act as templates for the editing process.

RNA editing of the transcript of the

cytochrome oxidase subunit II gene
from Trypanosoma brucei mitochondria.
(a) Insertion of 4 U residues (red)
produces a revised reading frame.

(b) A special class of guide RNAs,

complementary to the edited product,
act as templates for the editing process.

Note the presence of two G-U base

pairs, signified by a blue dot to indicate
non-Watson-Crick pairing.
 RNA editing

Deamination reactions that result in RNA

editing.

(a) The conversion of adenosine

nucleotides to inosine nucleotides is
catalyzed by ADAR enzymes - Adenosine
deaminases acting on RNA.

(b) Cytidine-to-uridine conversions are

catalyzed by the APOBEC family of
enzymes (apolipoprotein B mRNA editing
enzyme, catalytic polypeptide-like)

RNA editing of the transcript of the gene for the apoB100 component of LDL. Deamination,
which occurs only in the intestine, converts a specific cytidine to uridine, changing a Gln codon
to a stop codon and producing a truncated protein.
Transport of mRNA Across the Nuclear Envelope

Remodeling of mRNPs during

nuclear export.

Some mRNP proteins

(rectangles) dissociate from
nuclear mRNP complexes before
their export through an NPC.

Others (ovals) are exported

through the NPC with the mRNP,
but dissociate from it in the
cytoplasm and are shuttled back
into the nucleus through an NPC.

In the cytoplasm, translation

initiation factor eIF4E replaces
CBC bound to the 5′ cap, and
PABPC1 replaces PABPN1 (poly
A binding protein)
Reversible phosphorylation and direction of mRNP nuclear
export Step 1 : The yeast SR protein
Npl3 binds nascent pre-
mRNAs in its phosphorylated
form.
Step 2 : When polyadenylation
has occurred successfully, the
Glc7 nuclear phosphatase
dephosphorylates Npl3,
promoting the binding of the
mRNP exporter, NXF1/NXT1.
Step 3 : The mRNP exporter
allows diffusion of the mRNP
complex through the central
channel of the nuclear pore
complex (NPC).
Step 4 : The cytoplasmic
protein kinase Sky1
phosphorylates Npl3 in the
cytoplasm,
Followed by step 5 dissociation of the phosphorylated Npl3 from the mRNP exporter, probably
through the action of an RNA helicase associated with NPC cytoplasmic filaments
step 6 . The mRNA transporter and phosphorylated Npl3 are transported back into the nucleus
through NPCs.
Step 7 Transported mRNA is available for translation in the cytoplasm
Formation of heterogeneous ribonucleoprotein particles
(hnRNPs) and export of mRNPs from the nucleus.
(a) Model of a single chromatin transcription loop and
assembly of Balbiani ring (BR) mRNP in Chironomus
tentans. Nascent RNA transcripts produced from the
template DNA rapidly associate with proteins, forming
hnRNPs. The gradual increase in the size of the hnRNPs
reflects the increasing length of RNA transcripts at greater
distances from the transcription start site. The model was
reconstructed from electron micrographs of serial thin
sections of salivary gland cells.
(b) Schematic diagram of the biogenesis of hnRNPs.
Following processing of the pre-mRNA, the resulting
ribonucleoprotein particle is referred to as an mRNP.
(c) Model for the transport of BR mRNPs through the
nuclear pore complex (NPC) based on electron
microscopic studies. Note that the curved mRNPs appear
to uncoil as they pass through NPCs. As the mRNA enters
the cytoplasm, it rapidly associates with ribosomes,
indicating that the 5′ end passes through the NPC first
Summary of the composition and mass of ribosomes in bacteria
and eukaryotes
Ribosomal subunits are
identified by their S
(Svedberg unit) values,
sedimentation
coefficients that refer to
their rate of
sedimentation in a
centrifuge.

The S values are not

additive when subunits
are combined, because
S values are
approximately
proportional to the 2/3
power of molecular
weight and are also
slightly affected by
shape.
Processing of pre-rRNA transcripts in bacteria
1 Before cleavage, the 30S RNA precursor
is methylated at specific bases (red tick
marks), and some uridine residues are
converted to pseudo uridine (blue tick
marks) or dihydrouridine (black tick mark)
residues. The methylation reactions are of
multiple types, some occurring on bases
and some on 2’-hydroxyl groups.
2 Cleavage liberates precursors of rRNAs
and tRNA(s). Cleavage at the points
labeled 1, 2, and 3 is carried out by the
enzymes RNase III, RNase P, and RNase
E, respectively. RNase P is a ribozyme.
3 The final 16S, 23S, and 5S rRNA
products result from the action of a variety
of specific nucleases. The 7 copies of the
gene for pre-rRNA in the E. coli
chromosome differ in the number, location,
and identity of tRNAs included in the
primary transcript. Some copies of the
gene have additional tRNA gene segments
between the 16S and 23S rRNA segments
and at the far 3’ end of the primary
transcript
Processing of pre-rRNA transcripts in vertebrates
During transcription, the 45S primary transcript
is incorporated into a nucleolar 90S pre
ribosomal complex, in which rRNA processing
and ribosome assembly are tightly coupled.

1 The 45S precursor is methylated at more than

100 of its 14,000 nucleotides, either on the
bases or on the 2’-OH groups (red ticks), some
uridines are converted to pseudouridine (blue
ticks), and a few other modifications occur
(green ticks are dihydrouridine).

2 and 3 A series of enzymatic cleavages of the

45S precursor produces the 18S, 5.8S, and 28S
rRNAs, and the ribosomal subunits gradually
take shape with the assembling ribosomal
proteins.

The cleavage reactions and all of the

modifications require small nucleolar RNAs
(snoRNAs) found in protein complexes
(snoRNPs) in the nucleolus that resemble
spliceosomes. The 5S rRNA is produced
separately.
 Arrangement of genes that code tRNAs

Transfer RNAs – tRNA genes are located in small clusters scattered around the genome.

– tRNAs have promoter sequences within the coding region of the gene.

– During processing, the tRNA precursor is trimmed and numerous bases must be modified.
 Processing of tRNAs in bacteria and eukaryotes

The yeast tRNATyr (the tRNA specific for tyrosine binding;) is used to illustrate the important steps.

The nucleotide sequences shown in yellow are removed from the primary transcript. The ends are
processed first, the 5’ end before the 3’ end.

CCA is then added to the 3’ end, a necessary step in processing eukaryotic tRNAs and those
bacterial tRNAs that lack this sequence in the primary transcript.

While the ends are being processed, specific bases in the rest of the transcript are modified. For
the eukaryotic tRNA shown here, the final step is splicing of the 14 nucleotide intron. Introns are
found in some eukaryotic tRNAs but not in bacterial tRNAs.
 Outline
➢ Overview and interconnection of 4 molecular genetic processes

➢ Prokaryotic and eukaryotic translation

➢ The genetic code and history

➢ Recognition of codons by the tRNAs

➢ 5 major stages of protein synthesis

➢ Post translational modifications

➢ Assay designing for protein-DNA, protein-protein interactions

Overview of four basic molecular genetic processes
During transcription of a protein-coding
gene by RNA polymerase, the four-base
DNA code specifying the amino acid
sequence of a protein is copied, or
transcribed, into a precursor messenger
RNA (pre-mRNA) by the polymerization
of ribonucleoside triphosphate
monomers (rNTPs).
Removal of noncoding sequences and
other modifications to the pre-mRNA 2 ,
collectively known as RNA processing,
produce a functional mRNA, which is
transported to the cytoplasm.
During translation, the four-base code
of the mRNA is decoded into the 20–
amino acid language of proteins
During DNA replication 4, which occurs
only in cells preparing to divide,
deoxyribonucleoside triphosphate
monomers (dNTPs) are polymerized to
yield two identical copies of each
chromosomal DNA molecule. Each
daughter cell receives one of the
identical copies.
 Importance of protein synthesis
Proteins are the end products of most information pathways.

A typical cell requires thousands of different proteins at any given moment. These
must be synthesized in response to the cell’s current needs, transported
(targeted) to their appropriate cellular locations, and degraded when no longer
needed.

All the machinery are remarkably well conserved among life-forms from bacteria
to higher eukaryotes, denoting their presence in last universal common ancestor
(LUCA) of all extant organisms

About 300 different macromolecules cooperate to synthesize polypeptides,

understanding of which is greatest task in molecular biology

90% of the chemical energy used for protein synthesis by a cell, compared to all
biosynthetic reactions.

The 15,000 ribosomes, 100,000 molecules of protein synthesis–related protein

factors and enzymes, and 200,000 tRNA molecules in a typical bacterial cell can
account for more than 35% of the cell’s dry weight.

The study of protein synthesis offers another important reward: a look at a world
of RNA catalysts that may have existed before the dawn of life “as we know it.”
Prokaryotic translation Similarities Eukaryotic translation
5’ end of mRNA is immediately Occurs in cytoplasm Available only after post
available transcriptional modifications
mRNA is used as template
70S ribosomes = 50s+30S 80S ribosomes = 60S + 40S
(5S, 23S) + (16S) rRNA Ribosome is protein synthesis (5S, 5.8S, 28S) + (16S) rRNA
36 RNP + 21 RNP machinery 49 RNP + 33 RNP

Ribosomes present in cytoplasm 20 amino acids are common Ribosome attached to ER

Cap independent initiation 61 codon are similar Cap dependent and independent

SD sequences 8 nt up start sites Initiation (AUG, GUG or Only one start site at 5’ end
UUG/CUG), elongation,
Kozak sequence absent translocation and termination are Kozak at few NT upstream start
present site and help in initiation
30S recognize SD sequence in
mRNA Peptide bond formation process 40S recognize 5’ cap

1st tRNA is Met tRNAf – formyl Larger subunit of ribosome have 1st tRNA is met tRNA – met AA
met peptidyl transferase activity
7TFs eEF1 and eEf 2;
3 types of IF, Ef Tu and Ts; All 3 stop codons are similar termination by only 1 RF
termination by 3 RFs
20AA/sec Post translational modifications 1AA/sec
are present
“Understanding our universe requires hard work. At the same time,
no human endeavor is more exciting and potentially rewarding than
trying, and occasionally succeeding, to understand some part of the
natural world”
 History
When Whom What
1950s Paul Zamecnik Injected radio amino acids to mice and traced in liver proteins, when
hours passed by. Only in minutes they are found in ribosomes

1950s Francis Crick reasoning on how the genetic information encoded in the 4-letter
language of nucleic acids could be translated into the 20-letter language
of protein. Called Crick’s adaptor hypothesis
1950s Mahlon Hoagland Found that amino acids were “activated” when incubated with ATP and
and Zamecnik the cytosolic fraction of liver cells. Discovered tRNAs

1964 Robert Holley Heat stable RNA called tRNAs

1961 Marshall Nirenberg reported the first breakthrough. They incubated synthetic polyuridylate,
and Heinrich poly(U), with an E. coli extract, GTP, ATP, and a mixture of the 20
Matthaei radiolabeled aa in 20 different tubes. radioactive phenylalanine is
formed in 1 tube. In other words they synthesized homopolymers and
analysed translation.
1964 Nirenberg and ribosomes incubated with poly(U) and Phe-tRNAPhe bind both RNAs, but
Philip Leder if the ribosomes are incubated with poly(U) and some other aminoacyl-
tRNA, the aminoacyl-tRNA is not bound, because it does not recognize
the UUU triplets in poly(U)

1964 H. Gobind Khorana Synthesized polyribonucleotides with defined, repeating sequences of

two to four bases. Eg: threonine and histidine synthesized from
copolymer (AC)n, as it has alternating ACA and CAC codons:
ACACACACACACACA
The genetic code

Ribonucleoprotein particles or ribosomes were identified

as the site of protein synthesis.

Electron micrograph and schematic drawing of a portion

of a pancreatic cell, showing ribosomes attached to the
outer (cytosolic) face of the endoplasmic reticulum (ER).

The ribosomes are the numerous small dots bordering

the parallel layers of membranes

Crick reasoned that there must be an adaptor molecule

that facilitated translation of mRNA.
Today we know that the amino acid is covalently bound
at the 3’ end of a tRNA molecule and that a specific
nucleotide triplet elsewhere in the tRNA interacts with a
particular triplet codon in mRNA through hydrogen
bonding of complementary bases.
 Converting 4 letter code to 20 letter code

4 bases Vs. 20 amino acids

Case 1: 1 NT → 1 aa – only 4 aa will be covered

Case 2: 2 NT → 1 aa – only 16 aa will be covered

Case 3: 3NT → 1aa – all 20 aa will be covered

The genetic code – is it triplet… Overlapping? Non overlapping?
Several key properties of the genetic code were established in early genetic studies
Overlapping (O) versus nonoverlapping (N/o) genetic
codes.

In (N/o), codons (numbered consecutively) do not

share nucleotides.

In (O), some nucleotides in the mRNA are shared by

different codons. In a triplet code with maximum
overlap, many nucleotides, such as the third nucleotide
from the left (A), are shared by three codons.

Note that in (O), the triplet sequence of the first codon

limits the possible sequences for the second codon.

(N/o) provides much more flexibility in the triplet

sequence of neighboring codons and therefore in the
possible amino acid sequences designated by the
code.

The genetic code used in all living systems is now

known to be nonoverlapping
 The genetic code – experimental evidence
The triplet, nonoverlapping code:
Evidence for the general nature of the
genetic code came from many types of
experiments, including genetic
experiments on the effects of deletion and
insertion mutations.

Inserting or deleting one base pair alters

the sequence of triplets in a
nonoverlapping code; all amino acids
coded by the mRNA following the change
are affected.

Combining insertion and deletion

mutations affects some amino acids but
can eventually restore the correct amino
acid sequence. The triplet codons shaded in gray are those transcribed
from the original gene; codons shaded in blue are new
codons resulting from the insertion or deletion mutation
Adding or subtracting three nucleotides
leaves the remaining triplets intact,
providing evidence that a codon is triplet,
rather than four or five, nucleotides.
The question remined was: what were the
three-letter code words for each amino acid?
Homopolymers:

PolyA - Lys

PolyC - Pro

PolyU - Phe

PolyG – None – forms tetraplexes – codes for

glycene
 Remnant of Genetic code was cracked by
using artificial mRNA/ copolymer templates

i. Incorporation of AAA = 5/6 x 5/6 x 5/6 = 0.578

It can be normalized to 1.0 (0.578/0.578 = 1.0), based on relative frequency of occurrence

ii. The frequency of occurance of A2C = (5/6)(5/6)(1/6) = 0.116

Normalizing to AAA frequency of A2C = (0.116/0.578) = 0.2;
But A2C could be AAC, ACA and CAA so the overall frequency = 3x0.2

iii. Similarly expected frequency of AC2 = 0.04; overall frequency = 3x0.04

 Q. Determine expected frequency of polymer formation and tabulate if the A:C
ratio of 4:1 is used in polynucleotide polymerase reaction? (Normalize w.r.t AAA as
100)

Polymer Expected frequency Normalization to

assignment AAA occurrence
A2C 0.128 or 12.8% 0.25 or 25%
AC2 0.032 or 3.2% 0.0625 or 6.25%
AAA 0.512 or 51.2% 1 or 100%
CCC 0.008 or 0.8% 0.015 or 1.5%

i. Incorporation of AAA = 4/5 x 4/5 x 4/5 = 0.512

It can be normalized to 1.0 (0.512/0.512 = 1.0), based on relative frequency of occurrence

ii. The frequency of occurrence of A2C = (4/5)(4/5)(1/5) = 0.128

Normalizing to AAA frequency of A2C = (0.128/0.578) = 0.25;
But A2C could be AAC, ACA and CAA so the overall frequency = 3x0.25

iii. Similarly expected frequency of AC2 = 0.032; normalized = 0.0625, overall frequency =
3x0.0625
 What does these numbers mean???

If the co-polymer is made using different

stoichiometries of nucleotides, frequency of
occurrences of various combinations is different.

This frequency in turn determines frequency of

amino acid to be incorporated.

Polymer Expected frequency Normalization to

assignment AAA occurrence
A2C 0.128 or 12.8% 0.25 or 25%
AC2 0.032 or 3.2% 0.0625 or 6.25%
AAA 0.512 or 51.2% 1 or 100%
CCC 0.008 or 0.8% 0.015 or 1.5%
 Remnant of Genetic code was cracked by
using artificial mRNA/ copolymer templates

Summary of data from one of the early experiments designed to elucidate the genetic code. Poly A
polymerase is used here.

Incorporation of Thr, Asn, and Gln, His, Pro are values are very close to the expectation

When radiolabeled tRNAs were

used, frequency of incorporation
becomes “0” with non specific
trinucleotides.
Means aminoacyl tRNAs bind
specifically
But still the secret remains

Is this AAC or
ACA or CAA??

The remnant of genetic code was

cracked by artificially / chemically
synthesizing the codons and
understanding the incorporated
amino-acid by Har Gobind Khorana
 Three reading frames of genetic code

In a triplet, nonoverlapping code, all mRNAs have three potential reading frames, shaded here in
different colors. The triplets, and hence the amino acids specified, are different in each reading
frame.

Effect of a termination codon in a repeating tetranucleotide. Termination codons (orange) are

encountered every fourth codon in three different reading frames (shown in different colors).
Dipeptides or tripeptides are synthesized, depending on where the ribosome initially binds.
Based on the above, Dictionary” of
amino acid code words in mRNAs.

The codons are written in the 5’→3’

direction.

The third base of each codon (in

bold type) plays a lesser role in
specifying an amino acid than the
first two.

The three termination codons are

shaded in light red, the initiation
codon AUG in green.

All the amino acids except

methionine and tryptophan have
more than one codon.

In most cases, codons that specify

the same amino acid differ only at
the third base
A striking feature of the genetic code is that an amino acid may be specified by more than
one codon, so the code is described as degenerate.

This does not suggest that the code is flawed: although an amino acid may have two or more
codons, each codon specifies only one amino acid.

The degeneracy of the code is not uniform. Whereas methionine and tryptophan have single
codons, for example, three amino acids (Arg, Leu, Ser) have six codons, five amino acids
have four, isoleucine has three, and nine amino acids have two
 Wobble Allows Some tRNAs to Recognize More than One Codon

XYZ
X’ Y’ Z’

Pairing relationship of codon and

anticodon. (a) Alignment of the two
RNAs is antiparallel. The tRNA is
shown in the traditional cloverleaf
configuration. (b) Three different codon
pairing relationships are possible when
the tRNA anticodon contains inosinate
Wobble hypothesis gives an account of degeneracy of the genetic code

1. The first two bases of an mRNA codon always form strong Watson-Crick
base pairs with the corresponding bases of the tRNA anticodon and confer
most of the coding specificity.

2. The first base of the anticodon (reading in the 5’→3’ direction; this pairs
with the third base of the codon) determines the number of codons
recognized by the tRNA.
When the first base of the anticodon is C or A, base pairing is specific and
only one codon is recognized by that tRNA.
When the first base is U or G, binding is less specific and two different
codons may be read. When inosine (I) is the first (wobble) nucleotide of an
anticodon, three different codons can be recognized—the maximum number
for any tRNA. These relationships are summarized in Table 27–4.

3. When an amino acid is specified by several different codons, the codons

that differ in either of the first two bases require different tRNAs.

4. A minimum of 32 tRNAs are required to translate all 61 codons (31 to

encode the amino acids and 1 for initiation).
Q. Determine which amino acid should be attached to tRNAs with the following anticodons:

a) 5'-I-C-C-3'

b) 5'-G-A-U-3

5'-I-C-C-3’

3’-A-G-G-5’
3’-U-G-G-5’
3’-C-G-G-5’

5’-G-A-U-3’

3’-U-U-A-5’
3’-C-U-A-5’
Q. Determine which amino acid should be attached to tRNAs with the following anticodons:

a) 5’-I-A-G-3'

b) 5’-C-A-U-3’

5’-I-A-G-3’

3’-A-U-C-5’
3’-U-U-C-5’
3’-C-U-C-5’

5’-C-A-U-3’

3’-G-U-A-5’
Translation
The Ribosome Is a Complex Supramolecular Machine

First high-resolution structures of

bacterial ribosomal subunits by Thomas
Steitz, Ada Yonath, Venki
Ramakrishnan, Harry Noller, and others
– with their work focus in ribosomes is
shifted to rRNAs rather than proteins in
ribosomes

Summary of the composition and mass of

ribosomes in bacteria and eukaryotes.

Ribosomal subunits are identified by their S

(Svedberg unit) values, sedimentation
coefficients that refer to their rate of
sedimentation in a centrifuge.

The S values are not additive when subunits are

combined, because S values are approximately
proportional to the 2/3 power of molecular
weight and are also slightly affected by shape.
Transfer RNAs Have Characteristic Structural Features
General cloverleaf secondary structure of tRNAs.
The large dots on the backbone represent nucleotide
residues; the blue lines represent base pairs.
Characteristic and/or invariant residues common to all tRNAs
are shaded in light red.
Transfer RNAs vary in length from 73 to 93 nucleotides. Extra
nucleotides occur in the extra arm or in the D arm.
At the end of the anticodon arm is the anticodon loop, which
always contains seven unpaired nucleotides.
The D arm contains two or three D (5,6-dihydrouridine)
residues, depending on the tRNA.
In some tRNAs, the D arm has only three hydrogen-bonded
base pairs.
Symbols are: Pu, purine nucleotide; Py, pyrimidine
nucleotide; , pseudouridylate; G*, either guanylate or 2’-O-
methylguanylate

Three-dimensional structure of
yeast tRNAPhe deduced from x-ray
diffraction analysis by R. Holley.
Folding of cloverleaf into L
structure
General structure of aminoacyl-tRNAs.

The aminoacyl group is esterified to the 3’

position of the terminal A residue.

The ester linkage that both activates the amino

acid and joins it to the tRNA is shaded light red

Aminoacyl-tRNA synthetases - 20 enzymes, 1

per amino acid

Highly specific on BOTH business ends of the

tRNA:
i. Each must recognize several cognate tRNAs
Recognize several or all the tRNAs whose
anticodons complement the codons specifying a
particular amino acid

–Must recognize the correct amino acid

ii. Two different classes of aminoacyl-tRNA

synthetases, based on 3D structure
Stage 1: Aminoacyl-tRNA Synthetases
Attach the Correct Amino Acids in 2
steps
Also called tRNA charging

Step 1 is formation of an aminoacyl

adenylate, which remains bound to the
E-active site.

In step2 the aminoacyl group is

transferred to the tRNA. The mechanism
of this step is somewhat different for the
two classes of aminoacyl-tRNA
synthetases.

For class I enzymes, 2a the aminoacyl

group is transferred initially to the 2’-
hydroxyl group of the 3’-terminal A
residue, then 3a to the 3’-hydroxyl group
by a transesterification reaction.

For class II enzymes, 2b the aminoacyl

group is transferred directly to the 3’-
hydroxyl group of the terminal adenylate.

Convention - Met-tRNAMet
Stage 1: Aminoacyl-tRNA Synthetases
Attach the Correct Amino Acids –
proof reading occurs at this step

Aminoacylation of cognate tRNAs by synthetase

is based in part on greater affinities for these
types, coupled with weak affinities for
noncognate types.

In addition, non cognate tRNAs are unable to

fully undergo the induced-fit conformational
changes required for the later catalytic steps.
 Nucleotide positions in tRNAs that are
recognized by aminoacyl-tRNA synthetases
Some positions (purple dots) are the same in all tRNAs and
therefore cannot be used to discriminate one from another.

Other positions are known recognition points for one (orange) or

more (blue) aminoacyl-tRNA synthetases.

Structural features other than sequence are important for

recognition by some of the synthetases

Example: Structural elements of tRNAAla that are

required for recognition by Ala-tRNA synthetase.

(a) The tRNAAla structural elements recognized by the

Ala-tRNA synthetase are unusually simple.
A single G-U base pair (light red) is the only element
needed for specific binding and aminoacylation.
(b) A short synthetic RNA minihelix, with the critical G-U
base pair but lacking most of the remaining tRNA
structure.
b is aminoacylated specifically with alanine almost as
efficiently as the complete tRNAAla.
 Stage 2: A Specific Amino Acid Initiates Protein Synthesis

AUG initiation codon thus specifies an amino-terminal methionine

Exception: E. coli LacI protein, initiates with a GUG (Val) codon. In E. coli, AUG is the start codon
in approximately 91% of the genes, with GUG (7%) and UUG (2%) assuming this role more rarely.

Two tRNAs for methionine – i. for 5’ position (N-formylmethionyl-tRNAfMet); ii. for internal position
(tRNAMet)

Incorporation of 5’ a.a occurs two successive reactions, in prokaryotes

Methionine + tRNAfMet + ATP → Met-tRNAfMet + AMP + PP (E1)

N10 -Formyltetrahydrofolate + Met-tRNAfMet → tetrahydrofolate + fMet-tRNAfMet (E2)

The transformylase (E2) is more selective than the Met-tRNA synthetase (E1); it is specific for Met
residues attached to tRNAfMet
 Stage 2: Addition of amino acid residues to the carboxyl end

Proof that polypeptides grow by addition of amino acid residues to the carboxyl end: the
Howard Dintzis experiment.

Reticulocytes (immature erythrocytes) actively synthesizing hemoglobin were incubated with

radioactive leucine (selected because it occurs frequently in   - globin chains).

Samples of completed chains were isolated from the reticulocytes at various times afterward,
and the distribution of radioactivity was determined.
Stage 2: Addition of amino acid residues to the carboxyl end

The dark red zones show the portions of

completed  globin chains containing
radioactive Leu residues. At 4 min, only a
few residues at the carboxyl end of  globin
were labeled, because the only complete
globin chains with incorporated label after 4
min were those that had nearly completed
synthesis at the time the label was added.

With longer incubation times, successively

longer segments of the polypeptide
contained labeled residues, always in a
block at the carboxyl end of the chain.

The unlabeled end of the polypeptide (the

amino terminus) was thus defined as the
initiating end, which means that polypeptides
grow by successive addition of amino acids
to the carboxyl end
 Initiation of protein synthesis in prokaryotes.
Formation of the initiation complex in bacteria.
Blocking Ribosomal
The complex forms in three steps at the
subunit assembly expense of the hydrolysis of GTP to GDP and
Pi. IF-1, IF-2, and IF-3 are initiation factors.

50S + 30S contribute - P peptidyl site, A

aminoacyl site.; E exit site confined to 50S.

Complex consisting of the 30S ribosomal

subunit, IF-3, and mRNA is joined by both GTP-
bound IF-2 and the initiating fMet-tRNAfMet.

Here the anticodon of the tRNA is oriented 3’ to

5’, left to right, as in Figure 2 but opposite to the
orientation in below Figures
Messenger RNA sequences that serve as signals for initiation of protein synthesis in bacteria.

(a) Alignment of the initiating AUG (shaded in green) at its correct location on the 30S ribosomal
subunit depends in part on upstream Shine-Dalgarno sequences (light red).

Portions of the mRNA transcripts of five bacterial genes are shown. Exception: E. coli LacI protein,
which initiates with a GUG (Val) codon
In E. coli, AUG is the start codon in approximately 91% of the genes, with GUG (7%) and UUG
(2%) assuming this role more rarely.

(b) The Shine-Dalgarno sequence of the mRNA pairs with a sequence near the 3’ end of the 16S
rRNA
 Initiation of protein synthesis in eukaryotes.

The five steps.

Blocking- i. tRNA
binding to the A site
ii. Ribosomal binding Eukaryotic initiation
factors mediate the
association of first the
charged initiator tRNA to
form a 43S complex

mRNA (with the 5 cap

shown in red) to forms a
48S complex with the
help of eIF4G protein .

The final initiation

complex is formed as
the 60S subunit
associates, coupled with
the release of most of
the initiation factors.
 Initiation of protein synthesis in eukaryotes.
Eukaryotic mRNAs are bound to the ribosome as a complex with a number of specific binding
proteins.

Eukaryotic cells have at least 12 initiation factors. Initiation factors eIF1A and eIF3 are the
functional homologs of the bacterial IF-1 and IF-3, binding to the 40S subunit in step 1 and blocking
tRNA binding to the A site and premature joining of the large and small ribosomal subunits,
respectively.

The factor eIF1 binds to the E site. The charged initiator tRNA is bound by the initiation factor eIF2,
which also has bound GTP.

In step 2 this ternary complex binds to the 40S ribosomal subunit, along with two other proteins
involved in later steps, eIF5 (not shown in Fig) and eIF5B. This creates a 43S preinitiation complex.
The mRNA binds to the eIF4F complex, which,

In step 3, mediates its association with the 43S preinitiation complex. The eIF4F complex is made
up of eIF4E (binding to the 5’ cap), eIF4A (an ATPase and RNA helicase), and eIF4G (a linker
protein). The eIF4G protein binds to eIF3 and eIF4E to provide the first link between the 43S
preinitiation complex and the mRNA. The eIF4G also binds to the poly(A) binding protein (PABP) at
the 3’ end of the mRNA, circularizing the mRNA

In step 4, RNA helicase of eIF4A and another bound factor, eIF4B do the scanning function

In step 5, 60S ribosomal subunit associates with the complex, accompanied by the release of many
of the initiation factors. This requires the activity of eIF5 and eIF5B.
Circularization of the mRNA in the eukaryotic initiation complex

The 3’ and 5’ ends of eukaryotic mRNAs are

linked by the eIF4F complex of proteins.

The eIF4E subunit binds to the 5’ cap, and the

eIF4G protein binds to the poly(A) binding
protein (PABP) at the 3’ end of the mRNA.

The eIF4G protein also binds to eIF3, linking the

circularized mRNA to the 40S subunit of the
ribosome
Stage 3: Peptide Bonds Are Formed in the Elongation Stage : step 1
Binding of the second aminoacyl-tRNA

2nd aminoacyl-tRNA (AA2) enters the A site, bound

to GTP-bound EF-Tu (shown here as Tu).

It is accompanied by hydrolysis of the GTP and

release of the EF-Tu–GDP complex.

When the EF-Tu–GDP complex binds to EF-Ts,

bound GDP is released; EF-Ts is dissociated when
another molecule of GTP binds to EF-Tu.

This recycles EF-Tu and makes it available to

repeat the cycle.

2nd tRNA is accommodated with its conformation

pulls its aminoacyl end to P-site of previous tRNA.
Q. Formation of 1 peptide bond needs 18 k cal / mol energy; based on that calculate
energy needed for making a un deca peptide?

A. 180 kcal / mol

Q. 1 kilocalorie (kcal) of energy = 2.20 Celsius heat unit (CHU) based on this how
much heat is needed for formation of a un deca peptide?

A. 396OC
Stage 3: Step 2 –
peptide bond
formation

It require 18 k
cal/mol
energy;

1 kcal/mol = temp increase of 1oC in 1 Lit of water

The peptidyl transferase catalyzing this reaction is the 23S rRNA ribozyme.

2’-hydroxyl group of the 3’-terminal adenosine used as a general acid-base catalyst in this reaction

The N-fmet group (P) is transferred to the NH2 of the 2nd aminoacyl-tRNA (A site), forming a
dipeptidyl tRNA.

Both tRNAs bound to the ribosome shift position in the 50S subunit.

The uncharged and peptidyl tRNAs shifts so that their 3’ and 5’ ends are in the E and P sites, but
anticodons remain in the P and A sites.
 Stage 3: Step 3 - translocation.

Ribosome moves one codon 3’ direction of mRNA, with hydrolysis

energy of GTP bound to EF-G (translocase).

Dipeptidyl tRNA is now entirely in the P site; A site open for the
incoming (third) aminoacyl-tRNA.

Uncharged tRNA later dissociates from the E site, and the elongation
cycle begins again.

Structure of EF-G (with GDP) mimics

the structure of EF-Tu complexed with
tRNA.

The CTD of EF-G mimics the structure

of the anticodon loop of tRNA in both
shape and charge distribution.
The elongation cycle in eukaryotes is quite similar to that in bacteria.

Three eukaryotic elongation factors (eEF1, eEF1, and eEF2) have

functions analogous to those of the bacterial elongation factors (EF-Tu,
EF-Ts, and EF-G, respectively).

Eukaryotic ribosomes do not have an E site; uncharged tRNAs are

expelled directly from the P site
Stage 4: Termination of Polypeptide
Synthesis Requires a Special Signal

Occurs in response to a termination codon in the A site.

First, a release factor, RF (RF-1 or RF-2, depending on type

of termination codon), binds to the A site.

Hydrolysis of the ester linkage between the nascent

polypeptide and the tRNA in the P site is the result;
completed polypeptide released.

Finally, the mRNA, deacylated tRNA, and release factor

leave the ribosome, which dissociates into its 30S and 50S
subunits.

This is aided by ribosome recycling factor (RRF), IF-3, and

energy provided by EF-G–mediated GTP hydrolysis.

The 30S subunit complex with IF-3 is ready to begin another

cycle of translation
Stage 5: Newly Synthesized Polypeptide Chains Undergo Folding and
Processing
Nascent polypeptide chain is folded and processed into biologically active form

1D genetic message of mRNA is converted to 3D with Hydrogen, Hpbc, Van der walls forces

posttranslational modifications are set of changes to give bioactive confirmation to proteins

- They include i. Amino-Terminal and Carboxyl-Terminal Modifications - amino-terminal (formyl
met in prok and met in euk) residues may be removed in enzymatic reactions
- ii. 15 to 30 long signal sequences at amino-terminal end , that play protein targeting will be
removed by peptidases
- Modification of Individual Amino Acids

(a) Phosphorylated amino acids. (b) A carboxylated amino acid.

(c) Some methylated aa
- Carboxylation in glycoproteins - Asn residues (N-linked
oligosaccharides), in others to Ser or Thr residues (O-linked)

- Addition of Isoprenyl Groups

Stage 5: Newly Synthesized Polypeptide Chains Undergo Folding and
Processing

Farnesylation (isoprene units) of a Cys residue.

The thioether linkage is shown in red. The Ras
protein is the product of the ras oncogene. It is
important during signal transduction

- Addition of Prosthetic Groups – covalently bound forms Eg: biotin in acetyl-CoA

carboxylase; heme group of hemoglobin or cytochrome C

- Proteolytic Processing - trimming to form their smaller, active proteins eg: proinsulin, some
viral proteins, and proteases such as chymotrypsinogen and trypsinogen

- Formation of Disulfide Cross-Links (intrachain or interchain) between Cys residues - The

cross-links formed in this way help to protect the native conformation of the protein
molecule from denaturation in the extracellular environment, which can differ greatly from
intracellular conditions and is generally oxidizing.
 Protein synthesis summary

1 The tRNAs are aminoacylated.

2 Translation initiation occurs when an mRNA and an aminoacylated tRNA are bound to the
ribosome.
3 In elongation, the ribosome moves along the mRNA, matching tRNAs to each codon and
catalyzing peptide bond formation.
4 Translation is terminated at a stop codon, and the ribosomal subunits are released and recycled
for another round of protein synthesis.
5 Following synthesis, the protein must fold into its active conformation and ribosome components
are recycle
 Fidelity of protein synthesis
1st step that controls fidelity occurs during aminoacylation 1

The GTPase activity of EF-Tu during the first step of elongation in bacterial cells makes an
important contribution to the rate and fidelity of the overall biosynthetic process.
2
Both the EF-Tu–GTP and EF-Tu–GDP complexes exist for a few milliseconds before they
dissociate. These two intervals provide opportunities for the codon-anticodon interactions to be
proofread.

Incorrect aminoacyl-tRNAs normally dissociate from the A site during one of these periods. If the
GTP analog guanosine 5’-O-(3-thiotriphosphate) (GTPS) is used in place of GTP, slowed,
improving the fidelity (by increasing the proofreading intervals) but reducing the rate of protein
synthesis.

The process of protein synthesis has clearly been optimized through evolution to balance the
requirements for speed and fidelity.
 Fidelity of protein synthesis

Improved fidelity might diminish speed, whereas increases in speed would probably
compromise fidelity. Speed ∞ 1/ fidelity

And, recall that the proofreading mechanism on the ribosome establishes only that the proper
codon-anticodon pairing has taken place, not that the correct amino acid is attached to the
tRNA. 3

If a tRNA is successfully aminoacylated with the wrong amino acid (as can be done
experimentally), this incorrect amino acid is efficiently incorporated into a protein in response to
whatever codon is normally recognized by the tRNA.
 Leader sequences and protein targeting

Proteins destined for secretion, integration in the plasma membrane, or inclusion in lysosomes
generally share the first few steps in ER.

Mt, Cp and nuclear Proteins use three separate mechanisms.

Cytosolic proteins remain as it is

Günter Blobel and colleagues in 1970 postulated signal sequences

Amino-terminal signal sequences of some eukaryotic proteins that direct their translocation into
the ER.
The hydrophobic core (yellow) is preceded by one or more basic residues (blue).

Polar and short-side-chain residues immediately preceding (to the left of, as shown here) the
cleavage sites (indicated by red arrows).
Directing eukaryotic proteins with the appropriate signals to the ER
At NH3 ter – so Initiation on
synthesized free ribosomes
first

Recycled
SRP bound to SRP release follows
signal seq + GTP hydrolysis
ribosome

SRP directs to GTP Ribosome released

bound receptor

Signal sequence
cleaved

Elongation continues
in

This process involves the Signal recognition particle (SRP) cycle and translocation and
cleavage of the nascent polypeptide. George Palade demonstrated this.

SRP is a rod-shaped complex containing a 300 nucleotide RNA (7SL-RNA) and six different
proteins (combined Mr 325,000).
One protein subunit of SRP binds directly to the signal sequence, inhibiting elongation by
sterically blocking the entry of aminoacyl tRNAs and inhibiting peptidyl transferase. Another
protein subunit binds and hydrolyzes GTP.

The SRP receptor is a heterodimer of (Mr 69,000) and (Mr 30,000) subunits, both of which
bind and hydrolyze multiple GTP molecules during this process
Glycosylation of proteins in ER

Mimic UDP
N-acetyl All the proteins in ER
glucosamine
are modified by S-S
bonds, folding followed
by glycosylation.

N- linked glycosylation
for ER proteins; O-
linked for GC or
cytosol proteins
Directing eukaryotic secretion proteins, plasma membrane, or inclusion in
lysosomes

Pathway taken by proteins destined for

lysosomes, the plasma membrane, or
Phosphorylation of mannose residues on
secretion.
lysosome-targeted enzymes.
Proteins are moved from the ER to the cis side
N-Acetylglucosamine phosphotransferase
of the Golgi complex in transport vesicles.
recognizes some as yet unidentified
structural feature of hydrolases destined
Sorting occurs primarily in the trans side of the
for lysosomes.
Golgi complex
 Signal sequences that target proteins to different locations in bacteria

Basic amino acids (blue) near the amino terminus and hydrophobic core amino acids (yellow) are
highlighted.

The cleavage sites marking the ends of the signal sequences are indicated by red arrows.

OmpA is outer membrane protein A; LamB is a cell surface receptor protein for phage.
Model for secretory protein export in bacteria.
1 A newly translated polypeptide binds to the
cytosolic chaperone protein SecB,

2 delivered it to SecA, a protein associated

with the translocation complex (SecYEG) in
the bacterial cell membrane.

3 SecB is released, and SecA inserts itself

into the membrane, forcing about 20 amino
acid residues of the protein to be exported
through the translocation complex.

4 Hydrolysis of an ATP by SecA provides the

energy for a conformational change that
causes SecB to withdraw from the
membrane, releasing the polypeptide.

5 SecA binds another ATP, and the next

stretch of 20 amino acid residues is pushed
across the membrane through the
translocation complex.

Steps 4 and 5 are repeated until 6 the entire

protein has passed to the periplasm. The
electrochemical potential across the
membrane also provides some of the driving
force required for protein translocation