Chapter 13 Lecture

Concepts of Genetics Tenth Edition

The Genetic Code and Transcription

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Chapter 13 Contents

13.1 The Genetic Code Uses Ribonucleotide Bases as "Letters"

13.2 Early Studies Established the Basic Operational Patterns of the Code

13.3 Studies by Nirenberg, Matthaei, and Others Led to Deciphering of the Code

13.4 The Coding Dictionary Reveals Several Interesting Patterns among the 64 Codons

13.5 The Genetic Code Has Been Confirmed in Studies of Phage MS2

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Chapter 13 Contents

13.6 The Genetic Code Is Nearly Universal 13.7 Different Initiation Points Create

Overlapping Genes 13.8 Transcription Synthesizes RNA on a DNA

Template 13.9 Studies with Bacteria and Phages Provided

Evidence for the Existence of mRNA 13.10 RNA Polymerase Directs RNA Synthesis 13.11 Transcription in Eukaryotes Differs from

Prokaryotic Transcription in Several Ways

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Chapter 13 Contents

13.12 The Coding Regions of Eukaryotic Genes Are Interrupted by Intervening Sequences

13.13 RNA Editing May Modify the Final Transcript

13.14 Transcription Has Been Visualized by Electron Microscopy

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13.1 The Genetic Code Uses Ribonucleotide Bases as "Letters"

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• The genetic code is written in linear form, using the ribonucleotide bases that compose mRNA molecules as "letters"

• The sequence of RNA is derived from the complementary bases in the DNA

Section 13.1

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• In the mRNA, triplet codons specify one amino acid

• The code contains "start" and "stop" signals, certain codons (nonsense codons) that are necessary to initiate and to terminate translation

Section 13.1

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• The genetic code is – unambiguous – degenerate – commaless – nonoverlapping – nearly universal

Section 13.1

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13.2 Early Studies Established the Basic Operational Patterns of the Code

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• mRNA serves as an intermediate in transferring genetic information from DNA to proteins

Section 13.2

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• The triplet code provides 64 codons to specify the 20 amino acids

Section 13.2

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• The triplet nature of the code was revealed by frameshift mutations

Section 13.2

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• The genetic code reads three nucleotides at a time in a continuous, linear manner

• Thus, the code is nonoverlapping and commaless

Section 13.2

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• The genetic code is degenerate, which means that some amino acids are specified by more than one codon

• The nonsense codons do not specify an amino acid

Section 13.2

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13.3 Studies by Nirenberg, Matthaei, and Others Led to Deciphering of the Code

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• An in vitro protein synthesizing system along with the ability to produce synthetic mRNAs using polynucleotide phosphorylase provided the means for deciphering the genetic code

Section 13.3

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• Nirenberg and Matthaei added RNA homopolymers (RNA nucleotides with only one type of ribonucleotide) to the in vitro translation system to decipher which amino acids were encoded by the first few codons based on which amino acids were incorporated into the polypeptide

Section 13.3

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• Next, RNA heteropolymers (two or more different ribonucleosides) were used to decipher more codons employing the same method

Section 13.3

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• Nirenberg and Leder developed the triplet binding assay to determine other specific codon assignments

• In this technique, ribosomes bind to a single codon of three nucleotides, and the complementary amino acid-charged tRNA will be able to bind

Section 13.3

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• Long RNAs with di-, tri-, and tetranucleotide repeats were used for in vitro translation to determine more codon assignments

Section 13.3

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13.4 The Coding Dictionary Reveals Several Interesting Patterns among

the 64 Codons

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• The genetic code is degenerate, with many amino acids specified by more than one codon

• Only tryptophan and methionine are encoded by a single codon

Section 13.4

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• The wobble hypothesis predicts that the initial two ribonucleotides of triplet codes are often more critical than the third. The third position of the codon-anticodon interaction would be less spatially constrained and need not adhere as strictly to the established base-pairing rules at the third position of the codon

Section 13.4

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• The genetic code shows order in that chemically similar amino acids often share one or two middle bases in the triplets encoding them

Section 13.4

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• The initial amino acid incorporated into all proteins is a modified form of methionine—N-formylmethionine (fmet)

• AUG is the only codon to encode for methionine

• When AUG appears internally in mRNA, an unformylated methionine is inserted into the protein

Section 13.4

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• Three codons (UAG, UAA, and UGA) serve as termination codons and do not code for any amino acid

Section 13.4

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13.5 The Genetic Code Has Been Confirmed in Studies of Phage MS2

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• MS2 is a bacteriophage with only three genes on a 3500-base RNA genome

• Sequencing of the gene products confirmed the genetic code

• These genes specify a coat protein, an RNA-directed replicase, and a maturation protein

Section 13.5

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13.6 The Genetic Code Is Nearly Universal

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• Mitochondrial DNA revealed some exceptions to the universal genetic code

Section 13.6

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13.7 Different Initiation Points Create Overlapping Genes

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• In some viruses, overlapping genes (open reading frame, ORF) have been identified in which initiation at different AUG positions out of frame with one another leads to distinct polypeptides

Section 13.7

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13.8 Transcription Synthesizes RNA on a DNA Template

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• RNA is synthesized on a DNA template by the process of transcription

• The genetic information stored in DNA is transferred to RNA, which serves as the intermediate molecule between DNA and proteins

Section 13.8

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13.9 Studies with Bacteria and Phages Provided Evidence for the Existence

of mRNA

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• Analysis of RNA produced immediately after bacteriophage infection of E. coli shows that the base composition of the newly synthesized RNA resembles that of the phage DNA and not that of the bacterial host

• This suggests that RNA synthesis may be a preliminary step in protein synthesis

Section 13.9

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13.10 RNA Polymerase Directs RNA Synthesis

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• RNA polymerase directs the synthesis of RNA using a DNA template. No primer is required for initiation, and the enzyme uses ribonucleotides instead of deoxyribonucleotides. The reaction for RNA synthesis can be expressed as

n(NTP) (NMP)n + n(PPi) DNA

RNA polymerase

Section 13.10

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• RNA polymerase from E. coli contains the subunits α, β, β ′, ω, and σ

Section 13.10

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• Transcription begins with template binding by RNA polymerase at a promoter

• The σ subunit is responsible for promoter recognition (initiation of transcription)

Section 13.10

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• Transcription begins at the transcription start site, where the DNA double helix is unwound to make the template strand accessible to the action of RNA polymerase

• This site is called the transcription start site

Section 13.10

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• E. coli promoters have two consensus sequences, TTGACA and TATAAT (Pribnow box), positioned at –35 and –10 with respect to the transcription initiation site

• Mutations in any region diminish transcription, often severely

Section 13.10

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• Once initiation has been completed with the synthesis of the first 8–9 nucleotides, sigma (σ) dissociates and elongation proceeds under the direction of the core enzyme

Section 13.10

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• The enzyme traverses the entire gene until a termination nucleotide sequence is encountered

• In bacteria this termination is transcribed into RNA and causes the newly formed transcript to fold back on itself, forming what is called a hairpin structure held together by hydrogen bonds.

• In some cases, termination depends on the rho (ρ) termination factor

Section 13.10

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13.11 Transcription in Eukaryotes Differs from Prokaryotic Transcription in Several

Ways

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• Transcription in eukaryotes occurs in the nucleus and is not coupled to translation

Section 13.11

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• Eukaryotic transcription requires chromatin to become uncoiled, making the DNA accessible to RNA polymerase and other regulatory proteins. This transition is referred to as chromatin remodeling

Section 13.11

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• Eukaryotic RNA polymerases rely on transcription factors (TFs) to scan and bind to DNA

• In addition to promoters, enhancers and silencers also control transcription regulation

Section 13.11

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• Eukaryotic mRNAs require processing to produce mature mRNAs. – Addition of a 5′ cap – Addition of a 3′ tail – Excision of introns

Section 13.11

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• Eukaryotes possess three forms of RNA polymerase, each of which transcribes different types of genes

Section 13.11

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• RNA polymerase II (RNP II) is responsible for a wide range of genes in eukaryotes

• RNP II promoters have a core promoter element and promoter that determine where RNP II binds to the DNA and where it begins copying the DNA into RNA

Section 13.11

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Section 13.11

• The other three regulatory DNA sequences, proximal-promoter elements, enhancers, and silencers, influence the efficiency or rate of transcription initiation

• The TATA box is a core promoter element that binds the TATA-binding protein (TBP) of transcription factor TFIID and determines the start site of transcription

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• Enhancers and silencers – can be upstream, within, or downstream of

the gene – can modulate transcription from a distance – act to increase or decrease transcription in

response to cell's requirement for a gene product or at a particular time during development or place within an organism

Section 13.11

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• There are two broad categories of transcription factors that facilitate RNP II binding and initiation of transcription – General transcription factors are absolutely

required for all RNP II-mediated transcription – Transcription activators and repressors

influence the efficiency or the rate of RNP II transcription initiation

Section 13.11

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Section 13.11

• RNA polymerase opens up and separates (denatures) the two strands so that the template strand may pass through its active site during RNA synthesis

• As transcription proceeds, the enzyme moves along the DNA until the termination is encountered

• The enzyme-RNA complex separates

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• Heterogeneous nuclear RNA (hnRNA) is posttranscriptionally processed before it can leave the nucleus – Addition of a 5' cap that protects from

nuclease attack and may be involved in the transport of the transcript across the nucleus

– poly-A tail added to aid transport to cytoplasm – Introns are removed by splicing

Section 13.11

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13.12 The Coding Regions of Eukaryotic Genes Are Interrupted by Intervening

Sequences

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• Introns (intervening sequences) are regions of the initial RNA transcript that are not expressed in the amino acid sequence of the protein

Section 13.12

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• Introns are removed by splicing, and the exons are joined together in the mature mRNA

• The size of the mature mRNA is usually much smaller than that of the initial RNA

Section 13.12

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• Pre-mRNA introns are spliced out by the spliceosome in a reaction involving the formation of a lariat structure

Section 13.12

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13.13 RNA Editing May Modify the Final Transcript

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Section 13.13

• There are two main types of RNA editing prior to translation

• Substitution editing: the identities of individual nucleotide bases are altered; prevalent in mitochondria and chloroplast RNA derived in plants

• Insertion/deletion editing: nucleotides added/deleted from the total number of bases

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13.14 Transcription Has Been Visualized by Electron Microscopy

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Section 13.14

• Many RNA strands result from numerous transcription events occurring along each gene

• Ribosomes are attached to partially transcribed mRNA molecules and initiate translation

• Polyribosomes have been observed in both prokaryotes and eukaryotes

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