Download Nonsense-suppressing mutation causes addition of amino acid at

Chapter 8
Gene Expression
The Flow of Genetic Information
from DNA via RNA to Protein
Outline of Chapter 8

The genetic code


Transcription



How RNA polymerase, guided by base pairing, synthesizes a
single-stranded mRNA copy of a gene’s DNA template
Translation


How triplets of the four nucleotides unambiguously specify 20
amino acids, making it possible to translate information from a
nucleotide chain to a sequence of amino acids
How base pairing between mRNA and tRNAs directs the assembly
of a polypeptide on the ribosome
Significant differences in gene expression between
prokaryotes and eukaryotes
How mutations affect gene information and expression
The triplet codon represents each
amino acid

20 amino acids encoded for by 4 nucleotides

By deduction:
1 nucleotide/amino acid = 41 = 4 triplet combinations
 2 nucleotides/amino acid = 42 = 16 triplet
combinations
 3 nucleotides/amino acid = 43 = 64 triplet
combinations


Must be at least triplet combinations that code
for amino acids
The Genetic Code: 61 triplet codons represent 20
amino acids; 3 triplet codons signify stop
Fig. 8.3
A gene’s nucleotide sequence is colinear the amino
acid sequence of the encoded polypeptide




Charles Yanofsky – E. coli genes for a
subunit of tyrptophan synthetase compared
mutations within a gene to particular amino
acid substitutions
Trp- mutants in trpA
Fine structure recombination map
Determined amino acid sequences of
mutants
Fig. 8.4

A codon is composed of more than one
nucleotide
Different point mutations may affect same
amino acid
 Codon contains more than one nucleotide


Each nucleotide is part of only a single
codon

Each point mutation altered only one amino
acid
A codon is composed of three nucleotides and the starting
point of each gene establishes a reading frame
studies of frameshift mutations in bacteriophage T4 rIIB gene
Fig. 8.5


Fig. 8.6
Most amino acids
are specified by
more than one
codon
Phenotypic effect
of frameshifts
depends on if
reading frame is
restored
Cracking the code: biochemical manipulations
revealed which codons represent which amino acids

The discovery of messenger RNAs,
molecules for transporting genetic
information


Protein synthesis takes place in cytoplasm
deduced from radioactive tagging of amino
acids
RNA, an intermediate molecule made in
nucleus and transports DNA information to
cytoplasm
Synthetic mRNAs and in vitro translation determines which
codons designate which amino acids






1961 – Marshall Nirenberg
and Heinrich Mathaei
created mRNAs and
translated to polypeptides
in vitro
Polymononucleotides
Polydinucleotides
Polytrinucleotides
Polytetranucleotides
Read amino acid sequence
and deduced codons
Fig. 8.7

Fig. 8.8
Ambiguities
resolved by
Nirenberg and
Philip Leder using
trinucleotide
mRNAs of known
sequence to tRNAs
charged with
radioactive amino
acid with
ribosomes

5’ to 3’ direction of mRNA corresponds to N-terminal-to-Cterminal direction of polypeptide



One strand of DNA is a template
The other is an RNA-like strand
Nonsense codons cause termination of a polypeptide chain
– UAA (ocher), UAG (amber), and UGA (opal)
Fig. 8.9
Summary

Codon consist of a triplet codon each of which specifies an amino
acid





Codons are nonoverlapping
Code includes three stop codons, UAA, UAG, and UGA that
terminate translation
Code is degenerate
Fixed starting point establishes a reading frame



Code shows a 5’ to 3’ direction
UAG in an initiation codon which specifies reading frame
5’- 3’ direction of mRNA corresponds with N-terminus to Cterminus of polypeptide
Mutation modify message encoded in sequence



Frameshift mutaitons change reading frame
Missense mutations change codon of amino acid to another amino acid
Nonsense mutations change a codon for an amino acid to a stop codon
Do living cells construct polypeptides according to
same rules as in vitro experiments?


Fig. 8.10 a
Studies of how
mutations affect
amino-acid
composition of
polypeptides
encoded by a gene
Missense mutations
induced by
mutagens should be
single nucleotide
substitutions and
conform to the code


Proflavin treatment generates Trp- mutants
Further treatment generates Trp+
revertants

Single base insertion (Trp-) and a deletion
causes reversion (Trp+)
Fig. 8.10 b
Genetic code is almost universal but
not quite

All living organisms use same basic genetic
code
Translational systems can use mRNA from
another organism to generate protein
 Comparisons of DNA and protein sequence
reveal perfect correspondence between codons
and amino acids among all organisms

Transcription




RNA polymerase catalyzes transcription
Promoters signal RNA polymerase where to
begin transcription
RNA polymerase adds nucleotides in 5’ to 3’
direction
Terminator sequences tell RNA when to stop
transcription
Initiation of transcription
Fig. 8.11 a
Elongation
Fig. 8.11 b
Termination
Fig. 8.11 c
Information flow
Fig. 8.11 d
Promoters of 10 different bacterial genes
Fig. 8.12
In eukaryotes, RNA is processed
after transcription


A 5’ methylated cap and a 3’
Poly-A tail are added
Structure of the methylated
cap
How Poly-A tail is added to 3’ end of mRNA
Fig. 8.14
RNA splicing removes introns



Exons – sequences found in a gene’s DNA
and mature mRNA (expressed regions)
Introns – sequences found in DNA but not
in mRNA (intervening regions)
Some eukaryotic genes have many introns
Dystrophin gene underlying Duchenne muscular
dystrophy (DMD) is an extreme example of introns
Fig. 8.15
How RNA processing splices out
introns and adjoins adjacent exons
Fig. 8.16

Splicing is
catalyzed by
spliceosomes
Ribozymes –
RNA molecules
that act as
enzymes
 Ensures that all
splicing reactions
take place in
concert

Fig. 8.17

Alternative
splicing
Different mRNAs
can be produced
by same
transcript
 Rare transplicing
events combine
exons from
different genes

Fig. 8.18
Translation

Transfer RNAs (tRNAs) mediate translation of
mRNA codons to amino acids

tRNAs carry anticodon on one end


Structure of tRNA





Three nucleotides complementary to an mRNA codon
Primary – nucleotide sequence
Secondary – short complementary sequences pair and make
clover leaf shape
Tertiary – folding into three dimensional space shape like an L
Base pairing between an mRNA codon and a tRNA
anticodon directs amino acid incorporation into a
growing polypeptide
Charged tRNA is covalently coupled to its amino acid
Secondary and tertiary structure
Fig. 8.19 b
Aminoacyl-tRNA syntetase catalyzes attachment of
tRNAs to corresponding amino acid
Fig. 8.20
Base pairing between mRNA codon and tRNA anticodon
determines where incorporation of amino acid occurs
Fig. 8.21
Wobble:
Some tRNAs
recognize
more than
one codon for
amino acids
they carry
Fig. 8.22
Rhibosomes are site of polypeptide synthesis

Ribosomes
are complex
structures
composed of
RNA and
protein
Fig. 8.23
Mechanism of translation

Initiation sets stage for polypeptide synthesis


AUG start codon at 5’ end of mRNA
Formalmethionine (fMet) on initiation tRNA


Elongation during which amino acids are added to growing
polypeptide




First amino acid incorporated in bacteria
Ribosomes move in 5’-3’ direction revealing codons
Addition of amino acids to C terminus
2-15 amino acids per second
Termination which halts polypeptide synthesis


Nonsense codon recognized at 3’ end of reading frame
Release factor proteins bind at nonsense codons and halt
polypeptide synthesis
Initiation of translation
Fig. 8.24 a
Elongation
Fig. 8.24 b
Termination of translation
Fig. 8.24 c

Posttranslation
al processing
can modify
polypeptide
structure
Fig. 8.25
Significant differences in gene expression
between prokaryotes and eukaryotes


Eukaryotes, nuclear membrane prevents coupling of
transcription and translation
Prokaryotic messages are polycistronic


Eukaryotes, small ribosomal subunit binds to 5’
methylated cap and migrates to AUG start codon




Contain information for multiple genes
5’ untranslated leader sequence – between 5’ cap and AUG start
Only a single polypeptide produced from each gene
Initiating tRNA in prokaryotes is fMet
Initiating tRNA in eukaryotes is by unmodified Met.

Fig. 8.28
Nonsense
suppression

(a) Nonsense
mutation that
causes incomplete
nonfunctional
polypeptide

(b) Nonsensesuppressing
mutation causes
addition of amino
acid at stop codon
allowing production
of full length
polypeptide