PROTEIN

SYNTHESIS

Overview
A) TYPES OF RNA
B) TRANSCRIPTION
C) TRANSLATION
D) The lac OPERON

A) TYPES OF RNA

Three major types of RNA:

1.Messenger RNA
(mRNA)
- 3-5% of the
totalRNA
RNA
2. Transfer
(tRNA)
- 15% of the total RNA

3. Ribosomal RNA (rRNA)

- 80% of the total RNA

mRNA is single-stranded
formed on a single strand of
DNA by the process of
transcription in the nucleus

Base sequence of mRNA

is complementary to
that of DNA

CODON: is a set of three adjacent nucleotides, also

called triplet, in DNA or mRNA that designates a specific
amino acid to be incorporated into a polypeptide

mRNA exists for a short time

within a cell:
some minutes in
bacteria
Enzymes
degrade
mRNA

tRNA
Function: carry
amino acids present
in the cytoplasm to
the ribosome

tRNA
Structure:
the single
strand of a
tRNA molecule
is wound into a
double helix

All tRNA molecules

have the same basic
structure.

tRNA molecule
resembles a
clover leaf.

anticodo
n

5-end always
ends in guanine

The triplet base sequence at the

anticodon is directly related to the
amino acid carried by that tRNA
molecule.

3-end always ends

in CCA

Anticodon:
site of base pairing with mRNA
unique for each species of tRNA

How many types of tRNA?

About 45 different
kinds of tRNA
molecules
Why are there 45 and not
64 (one for each codon)?

As some tRNAs recognise more than

one codon due to the fact that the
third base-pair of a tRNA anticodon
allows some wobble
e.g. the alanine codons GCA, GCC,
and GCU are all recognised by the
same tRNA

GCA

GC C

codon

codon

G CU
codon

tRNA

rRNA
Synthesised by
genes inside
nucleolus called
nucleolar
organiser

The base sequence of rRNA is similar in

all organisms: prokaryotes & eukaryotes.

rRNA
Location of rRNA:
cytoplasm, associated with protein
molecules which together form
ribosomes
Function of ribosome:
polypeptide formation

Activation of tRNA requires:

1.energy from ATP
2.aminoacyl-tRNA
synthetase

Charged tRNA
molecules form

The activating enzymes

are highly specific.

The Central Dogma of molecular biology:

information flows in one direction

when genes are expressed

Exception to the Central

Dogma:
Viruses: acellular particles that reproduce
inside cells; many have RNA instead of
DNA.

Synthesis of DNA from RNA is:

reverse transcription.
Viruses that do this areretroviruses.
called:

Question: [MAY, 2011]

What is the Central Dogma of
genetics? (2)
DNA is transcribed to RNA
which is translated to protein.
Protein is never backtranslated to RNA or DNA;
And except for retroviruses,
DNA is never created from
RNA.

Overview
A) TYPES OF RNA
B)

TRANSCRIPTION
C) TRANSLATION
D) The lac OPERON

Two stages in protein synthesis:

making
mRNA from
Transcript
DNA
ion
Translatio
n base sequence in
mRNA is
translated into
an amino acid
sequence in a
protein

RNA Polymerase
catalyses synthesis of
RNA

Prokaryotes:
one RNA polymerase that
produces:
mRNA, tRNA and rRNA

Eukaryotes:
three different RNA polymerases
with distinct roles;
RNA polymerase II is responsible
for mRNA production [just
remember RNA polymerase]

Which strand in DNA is

transcribed?
Only one strand of DNA is
transcribedthe template
strand [3 to 5].

RNA is ANTIPARALLEL to the

DNA strand being transcribed
Why is the DNA template the 3 to
5 end strand?
TEMPLATE

RNA
polymerase

Nucleotides are
added ONLY at
the 3 end.

What is meant by nontemplate

strand?
The strand of DNA which is not
transcribed.

Transcription produces:
mRN
tRNA
rRNA
A

Promoter

Transcriptionunit

Initiationsite
RNApolymerase

Transcription
occurs in
three
phases:

DNA

Terminationsite

Initiation

TemplatestrandofDNA

Unwound

RNA

DNA

transcript

Rewound

2
Elongation

DNA

RNA
transcript

3
Termination

5
3

CompletedRNAtranscript

How does the cell know from where

to start transcription and where to
end?
About 20 000 genes in a human
cell!!

The promoter
a special sequence of DNA to which
RNA polymerase binds very tightly
the transcription of a gene begins at
the promoter

How many promoters in

a DNA?
There is at least one promoter for each
gene to be transcribed into mRNA.

Promoters serve as punctuation

marks, telling the RNA polymerase:

where to start

2
which strand of
DNA to read
3 the direction to

take from the start

A promoter, orients the RNA polymerase

and thus aims it at the correct strand
to use as a template

Promoter

Elongation: RNA polymerase unwinds

DNA about 10 base pairs at a time;
reads template in 3 to 5 direction.

RNA
polymerase

RNA polymerases do not

proofread and correct mistakes.

RNA
polymerase

RNA polymerase:
- opens the DNA strands
- synthesizes an RNA complementary to the
template

What is a nucleoside
triphosphate?

Termination: specified by a specific

DNA base sequence.
Mechanisms of termination are:
complex
varied

PostTranscriptio
nal
Modificatio
ns

Occur in
the
nucleus

The posttranscriptional
modifications involve:
1. modification of both
ends of pre-mRNA
2. removal of introns

1. Modification at both ends of

pre-mRNA

At the 5 end
a G-cap is added
(modified
guanosine
triphosphate)

At the 3 end
a poly A tail is
added
(100 to 300 adenine
nucleotides )

The G-cap
is added during transcription
role:
1. facilitates binding
to ribosome

2. protects it from
being digested by
ribonucleases

At 3 end a Poly A tail is added at

the end of transcription
In eukaryotes, there is
usually a polyadenylation
sequence (AAUAAA) near
the 3 end of the pre-mRNA,
after the last codon

AAUAAA:
acts as a signal
for an enzyme to
cut the pre-mRNA

Immediately after
this cleavage,
another enzyme adds
a poly A sequence

The poly A tail:

1. may assist in the export of the mRNA
from the nucleus
2. is important for the stability of mRNA

The posttranscriptional
modifications involve:
1. modification of both ends of
pre-mRNA

2. removal of
introns

Splicing to remove introns in

eukaryotes: occurs in the nucleus
to splice: to join ends together (Fig. 8)
exons - sections
that code for the
protein

Pre-mRNA

Mature-mRNA
[processed
mRNA]

introns - sections
that do not code
for the protein

Fig. 8 The spliceosome: an RNA splicing

machine.
Pre-mRNA is
bound by small
nuclear
ribonucleoprotein
particles (snRNPs)
(pronounced
snurps)
snRNPs cluster to
form a spliceosome

Explain why mRNA moving out of

the nucleus is shorter than that
produced during transcription.

Removal of introns.

Alternative splicing
exons can be linked together in different ways
70% of human genes are alternatively spliced
on average, a given gene gives rise
to4alternatively spliced variants - encoding a
total of90-100,000proteins which differ in their
sequence and therefore, in their activities

Allows for various proteins to be

formed from the same DNA fragment.

The diagram shows some of the steps involved

in the synthesis of insulin in a eukaryotic cell.

a) Where exactly
in the cell do
the following
processes take
place?
i) Transcription
Nucleus
ii) Translation
Cytoplasm

b) Suggest an organelle where

modification of the pro-insulin
polypeptide might occur.
Golgi apparatus

c)

Distinguish between introns

and exons.
Introns regions on DNA or RNA
that do not code for the protein
Exons regions on DNA or RNA
that code for the protein

d)Why is it important to modify the

mRNA after it is produced by
transcription in eukaryotic cells?

To remove the introns.

e) According to the figure above, what
does the stage further
Removal
modification involve?

of chain C.

Overview
A) TYPES OF RNA
B) TRANSCRIPTION

C)
TRANSLATION
D) The lac OPERON

Which organelle is concerned

with translation?

ribosome

What happens to
mRNA after the
polypeptide is
formed?

mRNA is
degraded
by enzymes.

Polyribosome
or Polysome:
several
ribosomes
attached to a
molecule of
mRNA

What is the advantage of polysomes?

Many polypeptides are synthesised
at the same time.

The Eukaryotic Ribosome Consists of:

small subunit :
1 rRNA
molecule
33 protein
molecules

A large subunit :
3 molecules of
rRNA

The ribosome subunits

assemble
during
translation.

A ribosome can combine with any mRNA and

all tRNAs. What is the significance of this?

A ribosome can be used to make

many different polypeptide
products.

Besides the cytoplasm, name TWO other regions

inside the eukaryotic cell that contain
ribosomes.

Chloroplast

Mitochondrion

Some ribosomes in chloroplast & mitochondria are

even smaller than those of prokaryotes.

FOUR sites on the large

subunit

tRNA

mRNA

Different books show different number

of sites
P & A sites needed for
syllabus

3 sites

2 sites

Translation
occurs on
ribosomes
in three
steps:
Ribosome moves
towards the 3 end

Start codon is AUG;

first amino acid is
always methionine,
which may be
removed after
translation.
The large subunit
joins the complex,
the charged tRNA is
now in the P site of
the large subunit.
Initiation factors are
responsible for
assembly of the
initiation complex.

Elongation: the second charged

tRNA enters the A site.

Large subunit catalyzes two reactions:

1

Breaks bond
between tRNA
in P site and its
amino acid
2

Peptide bond forms between

that amino acid and the amino
acid on tRNA in the A site

Because the large ribosomal

subunit performs these two actions,
it has peptidyl transferase activity

RNA acts as the catalyst it is

a ribozyme [normally
proteins are catalysts]
Why can RNA act
as a catalyst?

RNA can act as an enzyme as it can:

fold into 3D-shapes and
bind substrates, just as proteinbased enzymes do
Atomic structure of the large subunit.
Proteins are shown in blue and the two
RNA chains in orange and yellow.
The small patch of green in the center
of the subunit is the active site.

When the first tRNA

has released its
methionine, it
moves to the E site
and dissociates from
the ribosomecan
then become
charged again.
Elongation occurs as
the steps are
repeated, assisted
by proteins called
elongation factors.

Termination: translation ends

when a stop codon [UAA, UAG,
UGA] enters the A site.

Stop codon binds a

protein release
factorallows
hydrolysis of bond
between polypeptide
chain and tRNA on
the P site.

Polypeptide chain: C terminus is the

last amino acid added.

The ribosome
subunits
dissociate.

Enzymes
degrade mRNA

Translation

Large
subunit

P
Site

A
Site
mRNA

A U G
Small subunit

A C U U C G

Initiation
aa2

aa1

2-tRNA

1-tRNA
anticodon
hydrogen
bonds

U A C
A U G
codon

G A U
C

A C U U C G A
mRNA

Elongation

peptide bond
aa3
aa1

aa2

3-tRNA
1-tRNA
anticodon
hydrogen
bonds

U A C
A U G
codon

2-tRNA

G A A

G A U
C U A C U U C G A
mRNA

aa1

peptide bond
aa3
aa2

1-tRNA
3-tRNA

U A C
(leaves)

2-tRNA

A U G

G A A

G A U
C U A C U U C G A
mRNA

Ribosomes move over one codon

aa1

peptide bonds
aa4
aa2

aa3

4-tRNA
2-tRNA

A U G

3-tRNA

G C U

G A U G A A
C U A C U U C G A A C U
mRNA

peptide bonds

aa1

aa4

aa2
aa3

2-tRNA

4-tRNA

G A U
(leaves)

3-tRNA

A U G

G C U

G A A
A C U U C G A A C U
mRNA

Ribosomes move over one codon

peptide bonds

aa1

aa5

aa2
aa3

aa4

5-tRNA

U G A
3-tRNA

G C

4-tRNA

G A A G C U
A C U U C G A A C U
mRNA

peptide bonds

aa1

aa5

aa2
aa3

aa4

5-tRNA

U G A

3-tRNA

G A
G C

A
U

4-tRNA

C U

G C U
U C G A A C U
mRNA

Ribosomes move over one codon

aa4
aa3
aa2

aa5

Termination

aa199
aa200

primary
structure
of a protein

aa1
200-tRNA

AC
mRNA

C A

U G

terminator
or stop
codon

U U U A G

End Product
The end product of protein
synthesis is a primary
structure of a protein.
A sequence of amino acid
bonded together by peptide
bonds.
aa5
aa2
aa1

aa3

aa4

aa199
aa200

Translation
start
codon
mRNA

A U G G G C U C C A U C G G C G C A U A A
codon 1

protein methionine

codon 2

codon 3

glycine

serine

codon 4
isoleucine

codon 5

codon 6

glycine

alanine

codon 7
stop
codon

Primary structure of a protein

aa1

aa2

aa3
peptide bonds

aa4

aa5

aa6

Polypeptide folds
as it emerges
from the ribosome
What determines
how a protein folds?

The amino acid

sequence

Once a protein folds, how

will it know where to go?
The amino acid sequence
contains a signal
sequencean address
label

A protein may be moved to an

organelle or out of the cell

Post-translational
modifications

List four materials

(shown here) that are
required for translation
to occur:

1.
2.
3.
4.

Amino acids
mRNA
tRNA
ribosome

What happens if a mutation

occurs at a GENE?
Protein produced may fail to
function = genetic disease
e.g. Sickle-Cell Anaemia:
Mutation at a SINGLE base

Sickle-Cell Anaemia

 Haemoglobin
has more than
300 amino acids
A single base
change in the
gene for
haemoglobin
results in a lethal
condition

Wrong sequence of amino acids =

Wrong folding =
NON FUNCTIONAL PROTEIN

Essay Titles
1. Write an account about protein
synthesis in eukaryotic cells. [MAY, 2002]
2. Review the process of protein synthesis
in living systems. [MAY, 2008]
3. Trace the sequence of events through
which the information in a gene
eventually leads to the formation of a
protein. [MAY, 2009]

Overview
A) TYPES OF RNA
B) TRANSCRIPTION
C) TRANSLATION

D) The lac
OPERON

How did people first learn

about the mechanism by which
genes are regulated within the
cell?
From studies into the control of enzyme
synthesis in Escherichia coli

Bacteria were placed in a medium

having glucose and lactose. Explain
the growth curve obtained.

Glucose
used up

Lactose
used up

Why was there a period of time where

cells did not increase in number?

Enzymes were
being made to
metabolise lactose.

In E. coli enzymes can be synthesised:

1. continuously constitutive enzymes
2. only in the presence of an inducer
compound called inducible enzymes

Let us study how lactose

switches on genes so
that the enzymes
needed to metabolise
lactose are made

LACTOSE present:
Lactose is an INDUCER
(a compound that
stimulates synthesis of a
protein)

Enzymes are made

The proteins (enzymes)
produced are inducible
proteins

Growth on lactose requires

three
enzymes: permease
-galactosidase
-

is a carrier protein in the bacterial

plasma membrane that moves the lactose
into the cell

2. galactosidase
- breaks lactose

galactosi
dase
permeas
e

to glucose +
galactose

Growth on lactose requires

three enzymes:
3. -galactosidase
transacetylase
transfers an acetyl group from acetyl
coenzyme
A to certainin-galactosides
[galactoside
- a compound
-

which the H of the OH group on

carbon-1 of galactose is replaced
by an organic radical]
-

role of -galactosidase transacetylase in the

metabolism of lactose is not clear

How is it possible for

Prokaryotes to make
proteins only when they
are needed?
By transcriptional
regulation.

Jacob and Monod (in 1961)

proposed a mechanism to
explain how genes are
switched on and off in
bacteria

 In bacteria, genes are often

clustered into operons, composed
of:
An operator - an on-off switch
A promoter binds RNA
polymerase
Genes for metabolic enzymes
An operon can be switched off by
a protein called a repressor

Operons in E. coli
75 different operons controlling 250
structural genes have been identified
for E. coli
only the lac operon is in syllabus

The lac operon of E. coli

lacto
se

structural genes + the

stretches of DNA that
control their
transcription

The lac operon of E. coli

Part explained by Jacob
& Monod
Part
explained by other
scientists, later in time

The lac operon of E. coli

Promoter (plac)
binding site for
RNA

Operator (o)
binding site for
repressor

RNA polymerase cannot

bind to its promoter

No transcription

binds to its promoter

The following 7 slides refer to:

i

Promoter
Operator
Structuralgenes
Inducibilitygene
[Promoterforigene
isNOTshown]

Terminator

i
Transcription
mRNA

Translation

Repressor-protein

The
repressor
molecule is
made all
the time

LactoseAbsent
i

Repressor
molecule binds
to operator (O)

Repressor-protein

RNA
polymerase

Repressor-protein

RNA polymerase
cannot bind to
promoter (P)

Lactose binds to repressor

Lactose Present
i

mRNA

Repressor changes shape on binding to

lactose

RNA polymerase binds to

its promoter and
transcription occurs

Lactose Present
i

Translation results in
enzyme formation

mRNA is
polycistroni
c
so all 3
enzymes are
formed
together

SUMMARY
LACTOSE
absent

LACTOSE
present

Monocistronic vs polycistronic
mRNA
Polycistronic mRNA: in
prokaryotes
contains the genetic information to translate more
than one protein chain (polypeptide).

Monocistronic mRNA in
eukaryotes
contains the genetic information to
translate only a single protein chain
(polypeptide)

Question: [MAY, 2011]

Distinguish between the following
pairs of terms:

1.intron and exon; (2)

Intron is a sequence of nucleotides on
DNA or mRNA that does not code for the
protein.
Exon is a sequence of nucleotides on
DNA or mRNA that codes for the protein.

Question: [MAY, 2011]

2. post-transcriptional processing
and post-translational processing;
(2)
Post-transcriptional processing are
modifications that occur in mRNA after it
is released from DNA.
Post-translational processing are
modifications that occur on the
polypeptide after it is released from the
ribosome.

Question: [MAY, 2011]

3. codon and anti-codon. (2)
Codon is a sequence of bases in the DNA
or mRNA that code for an amino acid.
Anticodon is a sequence of bases in
tRNA which is complementary to the
codon on mRNA.

THE END