1 chapter of cof gnetics

8/8/2019 1 Chapter of Cof Gnetics

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Copyright 2009Pearson Education,Inc.

Art and Photos in PowerPoint

Concepts of GeneticsNinth Edition

Klug, Cummings, Spencer, Palladino

Chapter 14

The Genetic Code and Transcription


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).


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?





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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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)





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


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


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



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.



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


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




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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Copyright 2009Pearson Education,Inc. Table 14.1





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.





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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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


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 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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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.


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


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.


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.



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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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


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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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)


14.12 The Coding Regions of Eukaryotic

Genes Are Interrupted by Intervening

Sequences

Introns can be visualized by hybridizing mRNA withgenomic DNA.



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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.



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Sequences

14.12.1 Splicing Mechanisms: Autocatalytic RNAs

Self-splicing RNAs are rare, but they illustrate inimportant mechanism





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).



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


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 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.


14.13 Transcription Has Been Visualized by

Electron Microscopy

This demonstrated simultaneous translation (by multipleribosomes) and transcription in bacteria

Figure 14.15

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