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Concepts of GeneticsNinth Edition
Klug, Cummings, Spencer, Palladino
Chapter 14
The Genetic Code and Transcription
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Copyright 2009Pearson Education,Inc. Figure 14.1
The Central Dogma
Francis Crick suggested the
term CentralDogma 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).
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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
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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?
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14.2 Early Studies Established the Basic
Operational Patterns of the Code
14.2.2 The Nonoverlapping Nature of the Code
In 1954, George Gamow (1904-1968) suggested thatDNA controlled protein synthesis through tripletsof nucleotides.
Upon receiving a letter from Gamow, Crick admittedthat he and Watson hadnt even counted thenumber of amino acids.
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14.2 Early Studies Established the Basic
Operational Patterns of the Code
14.2.2 The Nonoverlapping Nature of the Code
Gamows suggested code was overlapping, but
Sydney Brenner(1927-) showed that the codecould not be overlapping.
He did this by showing that each amino acid had 19possible neighbors in proteins (Gamow did not
mind being proven wrong - in fact, he
communicated Brenners results to PNAS)
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14.2 Early Studies Established the Basic
Operational Patterns of the Code
14.2.2 The Nonoverlapping Nature of the Code
14.2.3 The Commaless and Degenerate Nature ofthe Code
Francis Crick (1916-2004) and Brennerestablishedthe triplet, commaless and degenerate nature ofthe genetic code using phage
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From Yanofsky (2007), adapted from Crick, F.H.C., L. Barnett, S. Brenner andR.J. Watts-Tobin (1961) Nature 192:12271232
14.2.3 The Commaless and Degenerate Nature of
the Code
Phage mutants that involved insertions or deletionswere made (the used proflavin, a chemical thatcauses indel mutations)
They found a mutant (named FC0) that they assumedto be a single base addition
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From Yanofsky (2007), adapted from Crick, F.H.C., L. Barnett, S. Brenner andR.J. Watts-Tobin (1961) Nature 192:12271232
14.2.3 The Commaless and Degenerate Nature ofthe Code
They found additional mutants that could produce awild-type phenotype upon recombination
Other mutants required two recombinations - thisreflects either the addition and subtraction of
one base or the addition of three bases
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14.2.3 The Commaless and Degenerate Nature ofthe Code
BIG IDEA for Crick et al. (1961) - if you have two
single-base indels in different directions (e.g. 1insertion and 1 deletion) they compensate foreach other. If you have three single-base indels
in the same direction (e.g. 3 insertions) they alsocompensate for each other.
The code must be degenerate - if there were only 20
functional codons out of 64 possible, you wouldnot see suppression in so many cases.
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14.2.3 The Commaless and Degenerate Nature ofthe Code
Crick et al. (1961) also showed that the genetic code
must be commaless. A code with commaswould 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 frameshiftmutations?
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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-FreeSystem
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 tothe NIH
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14.3 Studies by Nirenberg, Matthaei, andOthers Led to Deciphering of the Code
An RNA template for protein synthesis can be made
artificially using polynucleotide phosphorylase
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14.3 Studies by Nirenberg, Matthaei, and
Others Led to Deciphering of the Code
14.3.2 Homopolymer Codes
These would be poly(U), poly(A), etc.
14.3.3 Mixed Copolymers
Random mixtures of various nucleotides
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14.3 Studies by Nirenberg, Matthaei, and
Others Led to Deciphering of the Code
14.3.4 The Triplet Binding Assay
With Philip Leder, Nirenberg realized that triplets ofRNA are sufficient to stimulate ribosomeassembly. This allowed more codons to be
assigned.
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Copyright 2009Pearson Education,Inc.Figure 14-5 Copyright 2006 Pearson Prentice Hall, Inc.
Figure 14.5
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 r ibosomes bound to anitrocellulose filter.
Copyright 2009Pearson Education,Inc. Table 14.2
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14.3 Studies by Nirenberg, Matthaei, and
Others Led to Deciphering of the Code
14.3.5 Repeating Copolymers
Har Gobind Khorana (1922-), who shared the Nobel prizewith 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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Copyright 2009Pearson Education,Inc. Figure 14.6
Copyright 2009Pearson Education,Inc. Table 14.3
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14.3 Studies by Nirenberg, Khorana, Leder,Matthaei, and Others allowed the Codeto be Deciphered
By combining the data from all of the experiments
(Nirenberg-Matthaei, Nirenberg-Leder,Khoranas repeating copolymers) the geneticcode was filled in.
This code turns out to be virtually universal - allorganisms use the same code (or a minorvariant of the universal code)
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Copyright 2009Pearson Education,Inc.Figure 14-7 Copyright 2006 Pearson Prentice Hall, Inc.
Figure 14.7
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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
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14.4.1 Degeneracy and the Wobble Hypothesis
Wobble Hypothesis - proposed by Crick in 1966. He suggestedthat 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 allavailable 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 sayingthe preliminary evidence seems rather favourable tothe theory. I shall not be surprised if it proves correct.
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Copyright 2009Pearson Education,Inc. Table 14.4
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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 inbacteria]) and stop (UAA, UAG & UAG) codons.
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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.
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14.6 The Genetic Code Is Nearly Universal
Table 14.5
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14.7 Different Initiation Points Create
Overlapping Genes
Diagram showing the genes present in phage X174, a single-stranded DNA
phage sequenced in 1977 by Fred Sangerand colleagues.
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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, Franois 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 wereexpressed very rapidly.
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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.
Copyright 2009Pearson Education,Inc. Table 14.6
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14.10 RNA Polymerase Directs RNA
Synthesis
14.10.1 Promoters, Template Binding, and the
Subunit14.10.2 Initiation, Elongation, and Termination
of RNA Synthesis
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Copyright 2009Pearson Education,Inc. Figure 14.9
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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 ItsProcessing: Caps and Tails
Copyright 2009Pearson Education,Inc. Table 14.7
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, whichguide modifications of rRNA and snRNA).
RNA polymerase II (RNAP II orpol II) is the polymerase responsible fortranscription of mRNAs (technically, of hnRNA, the precursor or mRNA).
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Copyright 2009Pearson Education,Inc. Figure 14.10
Eukaryotic mRNAs are extensivelyprocessed
1) 5 capping - a 7 -methylguanosine
(modified G) is linked to the 5 end of
mRNAs through a p hosphotriesterbond.
2) Polyadenylation - the 3 end iscleaved and a poly(A) tail added.
3) Intron splicing - sequences withinthe hnRNA (heterogeneous nuclear
RNA, the term used for pre-mRNA)are removed
Almost all introns begin with GT (orGU,since we are describing RNA) and end
with AG (called the GT-AG rule)
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14.12 The Coding Regions of Eukaryotic
Genes Are Interrupted by Intervening
Sequences
Introns can be visualized by hybridizing mRNA withgenomic DNA.
Copyright 2009Pearson Education,Inc. Figure 14.11
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Copyright 2009Pearson Education,Inc. Figure 14.12
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14.12 The Coding Regions of EukaryoticGenes Are Interrupted by InterveningSequences
The notion of the cistronnow must be replaced by that of atranscription unit containing regions which will be lost from themature messenger - which I suggest we call introns (forintragenic regions) - alternating with regions that will beexpressed - exons. The gene is a mosaic: expressedsequences held in a matrix of silent DNA, an intronic matrix.The introns seen so far range from 10 to 10,000 bases inlength; I expect the amount of DNA in introns will turn out to befive to ten times the amount in exons. Walter Gilbert (1978)Why genes in pieces? Nature 271:501.
Copyright 2009Pearson Education,Inc. Table 14.8
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14.12 The Coding Regions of Eukaryotic
Genes Are Interrupted by Intervening
Sequences
14.12.1 Splicing Mechanisms: Autocatalytic RNAs
Self-splicing RNAs are rare, but they illustrate inimportant mechanism
Copyright 2009Pearson Education,Inc. Figure 14.13
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14.12 The Coding Regions of Eukaryotic
Genes Are Interrupted by Intervening
Sequences
14.12.2 Splicing Mechanisms: The Spliceosome
Most introns are spliced by the spliceosome, an RNA-
protein complex that includes snRNAs
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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 alternativespliceosome 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 limitednumber of protein coding genes (~20,000).
Copyright 2009Pearson Education,Inc. Figure 14.14
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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 consensusY-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
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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
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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 areexported to the cytoplasm to be degraded - this is an inside-outgene
Tycowski, K. T., Shu, M.-D., and Steitz, J.A. (1996) Nature 379:464-466.
Splicing ofproteins 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
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14.12 The Coding Regions of EukaryoticGenes Are Interrupted by InterveningSequences
14.12.3 RNA Editing Modifies the Final Transcript
There is extensive editing (insertion/deletion of Us) intrypanosomes (Trypanosoma brucei causes Africansleeping sickness)
Humans edit some transcripts - the apoB expressed in the smallintestine is edited, yielding a transcript that encodes aprotein about half as long as apoB in other tissues.
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14.13 Transcription Has Been Visualized by
Electron Microscopy
This demonstrated simultaneous translation (by multipleribosomes) and transcription in bacteria
Figure 14.15