Genetics - Lecture 3

How cells read the genome:

from RNA to protein
The genetic code
Three roles of RNA in translation
Translation initiation, elongation and
termination

How cells read the genome:

from RNA to protein
Intended Learning Outcomes
After completing the learning activities associated
with this lecture you should be able to:

1. Demonstrate a sound knowledge of the

principles of mRNA translation and protein
synthesis
2. Demonstrate a sound understanding of the
roles of RNA in translation

How cells read the genome:

from RNA to protein
During the lecture you should:
Take notes
Ask if you are puzzled

After the lecture you should:

Check you understand your notes
Consolidate your knowledge and understanding
by background reading

The "central dogma"

DNA
transcription

RNA
translation

protein

he central dogma (why does it matter?)

The structure and function of a cell depends on what proteins are produced, and in
what quantities
Differences in gene expression can depend on differences in both transcription and
translation
Cell type X:
Gene B

Gene A

Gene C

Gene D

transcription
none

some

some

lots

some

lots

some

C C C

D D

C C C

D D

RNA

translation

protein

The "central dogma"

DNA
Nucleus

transcription

RNA
Cytoplasm

translation

protein

Cracking the genetic code

How is information in nucleic
acids translated into proteins?
How is a sequence of bases
translated into a sequence of
amino acids?

Experiments to crack the genetic code

Francis Crick

Sydney Brenner

How many bases correspond to

one amino acid?
4 types of base in nucleic acids
20 types of amino acid in proteins
If 1 nt : a.a. then only 4 possible a.a.s
If 2 nt : a.a. then 42 = 16 possible a.a.s
If 3 nt : a.a. then 43 = 64 possible a.a.s
So:
There must be at least 3 nt per amino acid.
If all combinations used, code is
degenerate

Is the code overlapping?

No, because single base mutations
only ever affect one amino acid.

Crick, Brenner, et al.s experiment to

crack the genetic code
Used bacteriophage T4 which infects Escherichia coli:

Generated mutants in rII gene

Rapid lysis phenotype of larger plaques

Crick, Brenner, et al.s experiment to

crack the genetic code
Generated mutants in rII gene of bacteriophage
T4 using proflavin

planar molecule
intercalates between base pairs in DNA
causes frameshift mutations:
insertion of an extra base
or deletion of a base

Francis Cricks frameshift mutants in

rII gene of bacteriophage T4
FC0
FC1
FC2
FC3
FC4
FC5

(+) (+ = base insertion)

(-) (- = base deletion)
(+)
(-)
(+)
(-)

Double mutant
FC0 + FC1 (+ -)
FC1 + FC2 (- +)
FC0 + FC2 (+ +)
FC2 + FC3 (+ -)
FC1 + FC3 (- -)
FC0 + FC3 (+ -)

Phenotype
wt
wt
rIIwt
rIIwt
Bacteriophage
recombinants

Cricks hypothesis
Wild type:
THE BIG RED FOX ATE THE EGG
FC0 (+):
THE XBI GRE DFO XAT ETH EEG G
FC1 (-):
THE BIG RDF OXA TET HEE GG
FC0 + FC1 (+ -) pseudo-wild type:
THE XBI GRD FOX ATE THE EGG
(As expected double mutants of the same type are still mutant):
e.g. FC0 + FC2 (+ +):
THE XBI GYR EDF OXA TET HEE GG

Francis Cricks frameshift mutants in

rII gene of bacteriophage T4
FC0
FC1
FC2
FC3
FC4
FC5

(+) (+ = base insertion)

(-) (- = base deletion)
(+)
(-)
(+)
(-)

Carefully we double-checked the

numbers of the Petri dish to make sure
we had looked at the correct plate.
Everything was in order. I looked across
at Leslie. Do you realise, I said, that
you and I are the only people in the
world who know its a triplet code? .
Francis Crick

Double mutant
FC0 + FC1 (+ -)
FC1 + FC2 (- +)
FC0 + FC2 (+ +)
FC2 + FC3 (+ -)
FC1 + FC3 (- -)
FC0 + FC3 (+ -)

Phenotype
wt
wt
rIIwt
rIIwt

Triple mutant
FC0 + FC2 + FC4 (+ + +)
FC1 + FC3 + FC5 (- - -)
FC0 + FC1 + FC2 (+ - +)

Phenotype
wt(!)
wt(!)
rII(control)

(Double frameshift mutants

of the same type still have a
mutant phenotype)

Cricks hypothesis
Wild type:
THE BIG RED FOX ATE THE EGG
FC0 (+):
THE XBI GRE DFO XAT ETH EEG G
FC2 (+):
THE BIG YRE DFO XAT ETH EEG G
FC4 (+):
THE BIG RED ZFO XAT ETH EEG G
FC0 + FC2 + FC4 (+ + +) pseudo-wild type:
THE XBI GYR EDZ FOX ATE THE EGG

The genetic code of the mRNA is read in sets

of three nucleotides (the codons)

The genetic code was

deciphered in
biochemical experiments
by Marshall Nirenberg
and Har Gobind Khorana:
Robert Holley shared the
Nobel prize for his
discovery of tRNA.

The genetic code of the mRNA is read in sets

of three nucleotides (the codons)

5-3 codons

Amino acids

Some amino acids have more than one tRNA (redundancy/degeneracy)

Some tRNAs can tolerate a mismatch at the third codon position (wobble)
Three codons do not code for amino acids stop codons
Note the single-letter code for amino acids
this allows protein sequences to be analysed digitally bioinformatics

The genetic code is universal,

but many variations are known in mitochondria, and some in nuclear genomes

,
Met4

4
4

Candida species of yeast (including causative agents of candidiasis/thrush)

Micrococcus bacteria (Usp = loss of codon usage)
Ciliated protozoans and green algae
Vertebrate mitochondria
Mycoplasma bacteria (including human pathogens M. pneumoniae, M. genitalium)
Euplotes, a ciliate protozoan
Various organisms (SeC = the amino acid selenocysteine)
Spiroplasma bacteria
"Human tongue infected with oral candidiasis" by James Heilman

1
2,9
3
4
5
6
7
8

Lehman N (2001) Current Biology 11: R63 - R66

Which types of RNA participate in translation?

Type of RNA

Function

Messenger

mRNA

Encodes proteins

Ribosomal

rRNA

Forms part of the ribosome and catalyses protein

synthesis

Transfer

tRNA

Acts in protein synthesis as adaptors between

specific codon sequences on the mRNA and amino
acids

Small (nuclear,
nucleolar,
cytoplasmic)

snRNA,
snoRNA,
scRNA

Pre-mRNA processing, pre-rRNA processing,

transport of proteins to the ER and other cellular
processes

Micro

miRNA

Regulation of gene expression, by cleaving mRNAs

and inhibiting translation

Long non-coding

lncRNA

Many act as scaffolds for proteins; many regulatory

functions including X-chromosome inactivation

Other non-coding

ncRNA

RNA plays three roles in translation

B. tRNA
deciphers the
codons of the
mRNA

A. mRNA carries the genetic

information

C. rRNA makes
up the ribosome

tRNA

I. RNA plays three roles in translation

tRNA is the key to deciphering the codons in mRNA
1. Specific enzymes couple tRNAs to the correct amino acid

I. RNA plays three roles in translation

tRNA is the key to deciphering the codons in mRNA
1. Specific enzymes couple tRNAs to the correct amino acid

anticodon

2. The anti-codon in the charged tRNA pairs with the

codon in the mRNA, delivering its amino acid in the
correct sequence

. RNA plays three roles in translation

tRNA is the key to deciphering the codons in mRNA
The decoding process for translating nucleic acids sequence in mRNA into amino
acid sequence in proteins occurs in two steps:

1. Specific enzymes couple tRNAs to the correct amino acid

2. The anti-codon in the charged tRNA pairs with the codon in the
mRNA, delivering its amino acid in the correct sequence

. RNA plays three roles in translation

rRNA associates with a set of proteins to form the translational machinery
A ribosome is a large complex of four RNAs and more than 80 proteins

http://nobelprize.org/nobel_prizes/chemistry/laureates/2009/

. RNA plays three roles in translation

rRNA associates with a set of proteins to form the translational machinery
A ribosome is a large complex of four RNAs and more than 80 proteins

A-site = aminoacyl-tRNA site

P-site = peptidyl-tRNA site
E-site = exit site

Binding of tRNAs to the ribosome seen by cryo-EM

(From Valle et al., 2003 Nature Struct. Biol. 10, 899-906)

I. Translation can be broken down into three steps

INITIATION

In eukaryotes, translation usually initiates at the first (most 5) AUG codon in the
mRNA.

I. Translation can be broken down into three steps

INITIATION

I. Translation can be broken down into three steps

INITIATION

Initiator tRNA binds to

AUG start codon

The initiating AUG codon is bound by the initiator tRNAMet.

Therefore the first (amino-terminal) amino acid in the polypeptide will always be
methionine.

I. Translation can be broken down into three steps

INITIATION

Binding of the initiator tRNA to the small subunit allows the large subunit to bind
and the ribosome to assemble

I. Translation can be broken down into three steps

INITIATION

The P-site is filled with the initiator tRNA but the A-site is empty

I. Translation can be broken down into three steps

ELONGATION

The tRNA with an anticodon that corresponds to the codon in the A-site binds

I. Translation can be broken down into three steps

ELONGATION

The amino acid on the tRNA in the P-site is transferred onto the amino acid on the
tRNA in the A-site

I. Translation can be broken down into three steps

ELONGATION

The now empty tRNA moves to the exit site and is released. Now there is a
peptidyl tRNA in the A-site - what happens next?

I. Translation can be broken down into three steps

ELONGATION

The ribosome slides down the mRNA so that the tRNA with the growing peptide is
in the P-site, leaving the A-site free to bind another aminoacyl-tRNA

I. Translation can be broken down into three steps

ELONGATION

The growing peptide chain is transferred to the aminoacyl tRNA, converting it to a

peptidyl tRNA

I. Translation can be broken down into three steps

ELONGATION

And the cycle continues with the ribosome sliding, a new aminoacyl tRNA binding,
and peptidyl transfer followed by the exit of the empty tRNA

I. Translation can be broken down into three steps

ELONGATION

And the cycle continues with the ribosome sliding, a new aminoacyl tRNA binding,
and peptidyl transfer followed by the exit of the empty tRNA

I. Translation can be broken down into three steps

TERMINATION

At the end of the coding region of the mRNA there is a special STOP codon.
There are no tRNAs with anticodons that bind to STOP codons. Instead, STOP
codons are bound by termination factors

I. Translation can be broken down into three steps

TERMINATION

At the end of the coding region of the mRNA there is a special STOP codon.
There are no tRNAs with anticodons that bind to STOP codons. Instead, STOP
codons are bound by termination factors

I. Translation can be broken down into three steps

TERMINATION

At the end of the coding region of the mRNA there is a special STOP codon.
There are no tRNAs with anticodons that bind to STOP codons. Instead, STOP
codons are bound by termination factors

I. Translation can be broken down into three steps

TERMINATION

Translation ends with the mRNA and ribosomal subunits disassembling and the
release of the newly synthesised proteins

. RNA plays three roles in translation

B. tRNA
deciphers the
codons of the
mRNA

A. mRNA carries the genetic

information

C. rRNA makes
up the ribosome

Translation video:
http://www.dnalc.org/resources/3d/16-translation-advanced.html

mRNA is translated 5 to 3
Polypeptide synthesis is N to C
(amino-terminal to carboxy-terminal)

The ribosome is a ribozyme

Location of protein components (shown in gold)
of the bacterial large ribosomal subunit.
This view shows the interface with the small
ribosomal subunit. rRNA is shown in grey.
rRNA is responsible for the catalysis of
peptide bond formation.
Ban N et al. Science 289: 905-920

Genetic information flow

DNA
replication

DNA
transcription

RNA
translation

protein

Genetic information flow

DNA
replication

DNA
Information
carrier

transcription

RNA
Catalyst

(ribozymes)
translation

protein

RNA World?

Information
carrier

RNA
Catalyst

RNA World hypothesis

Ribozymes
Enzyme cofactors based on
nucleotides

(ribozymes)

RNA World hypothesis

DNA
replication

DNA
Information
carrier

reverse
transcription

transcription

RNA
Catalyst

(ribozymes)
translation

protein
RNA World hypothesis
Ribozymes
Enzyme cofactors based on
nucleotides

Genetic information flow

DNA
replication

DNA
reverse
transcription

transcription

RNA
translation

protein

Now please do this:

Check you understand your notes
Consolidate your knowledge and understanding by
background reading
Background reading
Relevant sections in Russell, or Campbell et al.
or relevant sections in any recent genetics, cell biology or
biochemistry textbook.
Translation video:
http://www.dnalc.org/resources/3d/16-translation-advanced.html

RNA World and the origins of life: End of Chapter 6 in:

Alberts B. et al. Molecular Biology of the Cell. 5th or 6th
Edition (2008/2015). Garland Science