1 Chapter of Cof Gnetics

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Concepts of Genetics
Ninth Edition
Klug, Cummings, Spencer, Palladino
Chapter 14
The Genetic Code and Transcription
Copyright © 2009©Pearson
Copyright Education,
2009 Pearson Inc.
Education, Inc.
The “Central Dogma”
Francis Crick suggested the

term “Central Dogma of
Molecular Biology” to describe
the pattern of information flow
in the cell in 1958.
The most prevalent processes

are transcription (DNA to RNA)
and translation (RNA to protein).
Copyright © 2009 Pearson Education, Inc. Figure 14.1
14.1 The Genetic Code Uses

Ribonucleotide Bases as “Letters”
There is a 1:1 relationship between DNA and RNA
DNA - A, C, G, and T RNA - A, C, G, and U
Copyright © 2009 Pearson Education, Inc.
1
14.2 Early Studies Established the Basic
Operational Patterns of the Code
14.2.1 The Triplet Nature of the Code
Amino acids are specified by triplets of nucleotides.

What does this mean?
How was this established?

14.2.2 The Nonoverlapping Nature of the Code

In 1954, George Gamow (1904-1968) suggested that
DNA controlled protein synthesis through triplets
of nucleotides.
Upon receiving a letter from Gamow, Crick admitted
that he and Watson hadn’t even counted the
number of amino acids.
2

Gamow’s suggested code was overlapping, but
Sydney Brenner (1927-) showed that the code
could not be overlapping.
He did this by showing that each amino acid had 19
possible neighbors in proteins (Gamow did not
mind being proven wrong - in fact, he
communicated Brenner’s results to PNAS)


14.2.3 The Commaless and Degenerate Nature of
the Code
Francis Crick (1916-2004) and Brenner established
the triplet, commaless and degenerate nature of
the genetic code using phage

the Code
Phage mutants that involved insertions or deletions
were made (the used proflavin, a chemical that
causes indel mutations)
They found a mutant (named FC0) that they assumed
to be a single base addition
From Yanofsky (2007), adapted from Crick, F.H.C., L. Barnett, S. Brenner and
R.J. Watts-Tobin (1961) Nature 192:1227–1232
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the Code
They found additional mutants that could produce a
wild-type phenotype upon recombination
Other mutants required two recombinations - this
reflects either the addition and subtraction of
one base or the addition of three bases
From Yanofsky (2007), adapted from Crick, F.H.C., L. Barnett, S. Brenner and
R.J. Watts-Tobin (1961) Nature 192:1227–1232

the Code
BIG IDEA for Crick et al. (1961) - if you have two

single-base indels in different directions (e.g. 1
insertion and 1 deletion) they compensate for
each other. If you have three single-base indels
in the same direction (e.g. 3 insertions) they also
compensate for each other.
The code must be degenerate - if there were only 20

functional codons out of 64 possible, you would
not see suppression in so many cases.

the Code
Crick et al. (1961) also showed that the genetic code

must be commaless. A code with commas
would look like:
… GAA X CTC X GCA X UAC X GUC …
Where ‘X’ is one or more bases that act as a “comma”,
separating the triplet codons.
Would a code with commas be sensitive to frameshift
mutations?
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14.3 Studies by Nirenberg, Matthaei, and
Others Led to Deciphering of the Code
14.3.1 Synthesizing Polypeptides in a Cell-Free

System
Marshall Nirenberg (1927-) pioneered the use of cell-
free translation systems
Nirenberg was a gator (‘48 B.S., ‘52 M.S.) who later
received a Ph.D. from Michigan and moved to
the NIH

An RNA template for protein synthesis can be made

artificially using polynucleotide phosphorylase

14.3.2 Homopolymer Codes

These would be poly(U), poly(A), etc.
14.3.3 Mixed Copolymers

Random mixtures of various nucleotides
5
Copyright © 2009 Pearson Education, Inc. Table 14.1

14.3.4 The Triplet Binding Assay

With Philip Leder, Nirenberg realized that triplets of
RNA are sufficient to stimulate ribosome
assembly. This allowed more codons to be
assigned.
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14.3.4 The Triplet Binding Assay
The Nirenberg-Leder experiment (published in 1964)
was based on measuring the binding of tRNA (or
sRNA as they called it) to ribosomes bound to a
nitrocellulose filter.

Figure 14-5 Copyright © 2006 Pearson Prentice Hall, Inc.

14.3.5 Repeating Copolymers

Har Gobind Khorana (1922-), who shared the Nobel prize
with Nirenberg in ‘68 for elucidating the genetic
code, used repeating copolymers.
Unlike the random mixtures used by Nirenberg, these
copolymers had a known sequence.
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14.3 Studies by Nirenberg, Khorana, Leder,

Matthaei, and Others allowed the Code
to be Deciphered
By combining the data from all of the experiments
(Nirenberg-Matthaei, Nirenberg-Leder,
Khorana’s repeating copolymers) the genetic
code was filled in.
This code turns out to be virtually universal - all
organisms use the same code (or a minor
variant of the universal code)
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Figure 14-7 Copyright © 2006 Pearson Prentice Hall, Inc.
14.4 The Coding Dictionary Reveals Several

Interesting Patterns among the 64
Codons
14.4.1 Degeneracy and the Wobble Hypothesis
Degeneracy - there is more than one codon for most

amino acids
14.4.1 Degeneracy and the Wobble Hypothesis
Wobble Hypothesis - proposed by Crick in 1966. He suggested

that 1st positions of tRNA anticodons (which bind the 3rd
position of codons) have relaxed base pairing rules.
Crick presented substantial evidence in favor of wobble, using all

available tRNA sequence data, the presence of the
modified tRNA base inosine (I), and the types of non-
sense suppressor mutants available.
Crick ended his wobble paper (J. Mol. Biol. 19:548) by saying
“…the preliminary evidence seems rather favourable to
the theory. I shall not be surprised if it proves correct.”
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14.4 The Coding Dictionary Reveals Several

Interesting Patterns among the 64
Codons
14.4.2 The Ordered Nature of the Code

Chemically similar amino acids group together.
14.4.3 Initiation, Termination, and Suppression
Specific codons act as start (AUG for Met [fMet in
bacteria]) and stop (UAA, UAG & UAG) codons.
14.5 The Genetic Code Has Been

Confirmed in Studies of Phage MS2
In 1976, the complete 3,569 bp sequence of the RNA

phage MS2 was determined.
The proteins had been sequenced independently, so it was
possible to find the genes encoding those proteins.
10
14.6 The Genetic Code Is Nearly Universal
14.7 Different Initiation Points Create

Overlapping Genes
Diagram showing the genes present in phage φX174, a single-stranded DNA

phage sequenced in 1977 by Fred Sanger and colleagues.
14.8 Transcription Synthesizes RNA on a

DNA Template
The nature of the code leaves open the mechanism by

which information moves from DNA to protein.
We now know that mRNA is responsible for this transfer of
information.
- An unstable but rapidly synthesized intermediate (like mRNA)
was postulated by Arthur Pardee, François Jacob, and Jacques
Monod based upon their “PaJaMo experiment”.
- PaJaMo (published in 1959 in the first volume of the Journal
of Molecular Biology) found that genes from Hfr strains were
expressed very rapidly.
11
14.9 Studies with Bacteria and Phages
Provided Evidence for the Existence of
mRNA
e.g., RNA produced during a phage infection hybridizes

with phage DNA. This indicates that the phage DNA
is the template for RNA.
14.10 RNA Polymerase Directs RNA

Synthesis
14.10.1 Promoters, Template Binding, and the

σ Subunit
14.10.2 Initiation, Elongation, and Termination
of RNA Synthesis
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14.11 Transcription in Eukaryotes Differs

from Prokaryotic Transcription in
Several Ways
14.11.1 Initiation of Transcription in Eukaryotes
14.11.2 Recent Discoveries Concerning RNA
Polymerase Function
14.11.3 Heterogeneous Nuclear RNA and Its
Processing: Caps and Tails
RNA Types are:

rRNA - ribosomal RNA mRNA - messenger RNA
tRNA - transfer RNA snRNA - small nuclear RNA
There are additional RNA types (e.g., snoRNA - small nucleolar RNAs, which
guide modifications of rRNA and snRNA).
RNA polymerase II (RNAP II or pol II) is the polymerase responsible for

transcription of mRNAs (technically, of hnRNA, the precursor or mRNA).
13
Eukaryotic mRNAs are extensively
processed
1) 5’ capping - a 7-methylguanosine
(modified G) is linked to the 5’ end of
mRNAs through a phosphotriester
bond.
2) Polyadenylation - the 3’ end is
cleaved and a poly(A) tail added.
3) Intron splicing - sequences within
the hnRNA (heterogeneous nuclear
RNA, the term used for pre-mRNA)
are removed
Almost all introns begin with GT (or GU,

since we are describing RNA) and end
with AG (called the GT-AG rule)
14.12 The Coding Regions of Eukaryotic

Genes Are Interrupted by Intervening
Sequences
Introns can be visualized by hybridizing mRNA with

genomic DNA.
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Sequences
“The notion of the cistron…now must be replaced by that of a
transcription unit containing regions which will be lost from the
mature messenger - which I suggest we call introns (for
intragenic regions) - alternating with regions that will be
expressed - exons. The gene is a mosaic: expressed
sequences held in a matrix of silent DNA, an intronic matrix.
The introns seen so far range from 10 to 10,000 bases in
length; I expect the amount of DNA in introns will turn out to be
five to ten times the amount in exons.” Walter Gilbert (1978)
Why genes in pieces? Nature 271:501.
15
Sequences
14.12.1 Splicing Mechanisms: Autocatalytic RNAs
“Self-splicing” RNAs are rare, but they illustrate in

important mechanism

Sequences
14.12.2 Splicing Mechanisms: The Spliceosome
Most introns are spliced by the spliceosome, an RNA-

protein complex that includes snRNAs
16
The GT-AG (or GU-AG) Rule
Intron boundaries are defined by the nucleotides GU (GT

in DNA) and AG.
Called the GT-AG rule.
Splicing enhancers (and silencers) are found in the exons.
The majority of animal and plant introns are removed by the spliceosome that
recognizes GT-AG introns.
However, plants and animals (but not fungi) have a second “alternative”
spliceosome that is responsible for splicing non-canonical introns.
After removal from the primary transcript, virtually all introns are degraded.
Alternative splicing may explain the complexity of vertebrates despite our limited
number of protein coding genes (~20,000).
Co-Transcriptional RNA Processing
The RNA polymerase II C-terminal domain (CTD) can be

phosphorylated and it binds to enzymes involved in RNA
processing.
This included both addition of the 5’ cap and the splicing of introns.
The basic CTD sequence is repeat with the consensus Y-S-P-T-S-P-S
The CTD is absent or very different in some putatively primitive eukaryotes (e.g.,
Giardia, Trichomonas, trypanosomes).
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A single gene can have multiple splice forms
RNA Processing - Alternative Splicing

The fact that some introns are spliced only under specific
conditions provides another way to regulate genes.
For example, sex determination in Drosophila results from a
set of genes with alternatively spliced introns.
e.g., intron 3 in the Sxl (sex lethal) gene of female Drosophila is skipped
“Inside-Out” Genes and Inteins
In most genes, the introns are removed and degraded while

the EXON sequences are functional.
The snoRNAs are involved in ribosome assembly (small
nucleolar RNAs).
One snoRNA (called U22) is present in an intron and the exons are
exported to the cytoplasm to be degraded - this is an “inside-out”
gene
Tycowski, K. T., Shu, M.-D., and Steitz, J.A. (1996) Nature 379:464-466.
Splicing of proteins has also been described.
These proteins are called INTEINS.
Inteins are autocatalytically removed from the spliced EXTEIN
sequences.
Surprisingly, inteins are related to nucleases.
More information on inteins can be found at:
http://www.neb.com/neb/inteins.html
18
Sequences
14.12.3 RNA Editing Modifies the Final Transcript

There is extensive editing (insertion/deletion of U’s) in
trypanosomes (Trypanosoma brucei causes African
sleeping sickness)
Humans edit some transcripts - the apoB expressed in the small
intestine is edited, yielding a transcript that encodes a
protein about half as long as apoB in other tissues.
14.13 Transcription Has Been Visualized by

Electron Microscopy
This demonstrated simultaneous translation (by multiple
ribosomes) and transcription in bacteria
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Art and Photos in PowerPoint®

The “Central Dogma”

Francis Crick suggested the

The most prevalent processes

Copyright © 2009 Pearson Education, Inc. Figure 14.1

14.1 The Genetic Code Uses

There is a 1:1 relationship between DNA and RNA

DNA - A, C, G, and T RNA - A, C, G, and U

Copyright © 2009 Pearson Education, Inc.

14.2.1 The Triplet Nature of the Code

Amino acids are specified by triplets of nucleotides.

Copyright © 2009 Pearson Education, Inc.

Copyright © 2009 Pearson Education, Inc. Figure 14.2

14.2 Early Studies Established the Basic

14.2.2 The Nonoverlapping Nature of the Code

Copyright © 2009 Pearson Education, Inc.

14.2.2 The Nonoverlapping Nature of the Code

14.2 Early Studies Established the Basic

14.2.2 The Nonoverlapping Nature of the Code

Copyright © 2009 Pearson Education, Inc.

14.2.3 The Commaless and Degenerate Nature of

14.2.3 The Commaless and Degenerate Nature of

BIG IDEA for Crick et al. (1961) - if you have two

The code must be degenerate - if there were only 20

Copyright © 2009 Pearson Education, Inc.

14.2.3 The Commaless and Degenerate Nature of

Crick et al. (1961) also showed that the genetic code

Copyright © 2009 Pearson Education, Inc.

14.3.1 Synthesizing Polypeptides in a Cell-Free

Copyright © 2009 Pearson Education, Inc.

14.3 Studies by Nirenberg, Matthaei, and

An RNA template for protein synthesis can be made

Copyright © 2009 Pearson Education, Inc. Figure 14.3

14.3 Studies by Nirenberg, Matthaei, and

14.3.2 Homopolymer Codes

14.3.3 Mixed Copolymers

Copyright © 2009 Pearson Education, Inc.

Copyright © 2009 Pearson Education, Inc. Figure 14.4

14.3 Studies by Nirenberg, Matthaei, and

14.3.4 The Triplet Binding Assay

Copyright © 2009 Pearson Education, Inc.

Copyright © 2009 Pearson Education, Inc. Figure 14.5

Copyright © 2009 Pearson Education, Inc. Table 14.2

14.3 Studies by Nirenberg, Matthaei, and

14.3.5 Repeating Copolymers

Copyright © 2009 Pearson Education, Inc.

Copyright © 2009 Pearson Education, Inc. Table 14.3

14.3 Studies by Nirenberg, Khorana, Leder,

Copyright © 2009 Pearson Education, Inc.

14.4 The Coding Dictionary Reveals Several

14.4.1 Degeneracy and the Wobble Hypothesis

Degeneracy - there is more than one codon for most

Copyright © 2009 Pearson Education, Inc.

14.4.1 Degeneracy and the Wobble Hypothesis

Wobble Hypothesis - proposed by Crick in 1966. He suggested

Crick presented substantial evidence in favor of wobble, using all

Copyright © 2009 Pearson Education, Inc.

14.4 The Coding Dictionary Reveals Several

14.4.2 The Ordered Nature of the Code

Copyright © 2009 Pearson Education, Inc.

14.5 The Genetic Code Has Been

In 1976, the complete 3,569 bp sequence of the RNA

Copyright © 2009 Pearson Education, Inc.