Download The nucleotide sequence of a gene is colinear with the amino acid

A 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 64 triplet combinations that
code for 20 amino acids
The Genetic Code: 61 triplet codons represent 20
amino acids; 3 triplet codons signify stop
Fig. 8.3
The nucleotide sequence of a gene is colinear with
the amino acid sequence of the polypeptide




Charles Yanofsky – compared mutations
within a gene to particular amino acid
substitutions
Trp- mutants in the trpA gene that encodes
tryptophan synthetase
Fine structure recombination map
Determined amino acid sequences of
mutants
Fig. 8.4
Yanofsky’s conclusions

A codon is composed of more than one
nucleotide


Different point mutations may affect same
amino acid
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
reading frame
Cracking the code: biochemical manipulations
revealed which codons represent which amino acids

The discovery of messenger RNAs
Protein synthesis takes place in cytoplasm
deduced from radioactive tagging of amino
acids
 An intermediate molecule made in nucleus 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 in vitro
Polymononucleotides
Polydinucleotides
Polytrinucleotides
Polytetranucleotides
Determined amino acid
sequence to deduce codons
Fig. 8.7

Fig. 8.8
Ambiguities
resolved by
Nirenberg and
Philip Leder using
trinucleotide
mRNAs of known
sequence and
tRNAs charged
with a radioactive
amino acid


5’ to 3’ direction of mRNA corresponds to N-terminal-to-Cterminal direction of polypeptide
Nonsense codons cause termination of a polypeptide chain
– UAA (ochre), UAG (amber), and UGA (opal)
Fig. 8.9
Do living cells construct polypeptides according to
same rules as in vitro experiments?
Fig. 8.10 a

How gene mutations
affect amino-acid
composition

Missense mutations
should 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 correspondence between codons and
amino acids among all organisms

Specialized
example of
regulation
through
RNA
stability
Fig. 17.17
Promoters of 10 different bacterial genes
Fig. 8.12
Regulatory elements that map near a
gene are cis-acting DNA sequences

cis-acting elements


Promoter – very close to initiation site
Enhancer



Can be far way from gene
Can be in either orientation
Function to augment or repress basal levels of transcription
Fig. 17.1 a
Fig. 16.2
In eukaryotes three RNA polymerases
transcribe different sets of genes

RNA polymerase I
transcribes rRNA


Fig. 17.2 a
rRNAs are made of
tandem repeats on
one or more
chromosomes
RNA polymerase I
transcribes one
primary transcript
which is broken
down into 28s and
5.8s by processing

Fig. 17.2 b
RNA polymerase III transcribes
tRNAs and other small RNAs (5s
rRNA, snRNAs)

RNA polymerase II transcribes all protein
coding genes
Reporter constructs are a tool for
studying gene regulation



Sequence of DNA containing regulatory
region, but not coding region
Coding region replaced with easily
identifiable product
In vitro mutagenesis can be used to
systematically alter the presumptive
regulatory region
Fusion used to perform genetic studies of the
regulatory region of gene X
Fig. 16.18 a
Creating a
collection of
lacZ
insertions in
the
chromosome
Fig. 16.18 b
Use of a fusion to
overproduce a gene
product
Fig. 16.18 c
Reporter constructs in worms
Fig. C.8
GFP tagging can be used to follow the
localization of proteins


Fig. 19.18 c,d
Recombinant gene
encoding a GFP
fusion protein at C
terminus
Mouse with GFPlabeled transgene
expressed
throughout body
Enhancer trapping to identify genes
by expression pattern





P element with lacZ gene
downstream of promoter
When mobilized, 65% of
new insertions express lacZ
reporter during
development
Promoter can only activate
transcription if under
control of enhancers of
genes near insertion site
Detects genes turned on in
certain tissues
Genes isolated by plasmid
rescue
Fig. D.10
Regulatory elements that map far from a
gene are trans-acting DNA sequences

Proteins that
interact directly or
indirectly with cisacting elements


Transcription
factors
Identified by:

Fig. 17.1 b
Biochemical studies
to identify proteins
that bind in vitro to
cis-acting elements
trans-acting proteins control transcription
from class II promoters

Basal factors bind to
the promoter
TBP – TATA box
binding protein
 TAF – TBP
associated factors


Fig. 17.4 a
RNA polymerase II
binds to basal
factors
Most regulatory proteins are
oligomeric
 More than one
binding domain
DNase footprint
identifies binding
region
DNase cannot
digest protein
covered sites
Fig. 16.15 a
Activating factors



Bind to enhancer DNA in specific ways
Interact with other proteins to activate and
increase transcription as much as 100-fold
above basal levels
Two structural domains mediate these
functions
DNA-binding domain
 Transcription-activator domain


Transcriptional
activators bind
to specific
enhancers at
specific times to
increase
transcriptional
levels
Fig. 17.5 a
Examples of common transcription factors

helix-loophelix and
zinc-finger
proteins bind
to the DNA
binding
domains of
enhancer
elements
Fig. 17.5 b
Leucine zipper – a common activator protein
with dimerization domains
Fig. 17.7 b
Some eukaryotic activators must
form dimers to function

Eukaryotic transcription factor protein structure


Homomers – multimeric proteins composed of identical
subunits
Heteromers – multimeric proteins composed of
nonidentical subunits
Fig. 17.7 a
Localization of activator domains
using recombinant DNA constructs



Fig. 17.6
Fusion constructs
from three parts of
gene encoding an
activator protein
Reporter gene can
only be transcribed
if activator domain
is present in the
fusion construct
Part B contains
activation domain,
but not part A or C