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Chapter 8 Gene Expression. The Flow of Genetic Information from DNA via RNA to Protein. Outline of Chapter 8. The genetic code

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Chapter 8 Gene Expression

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Chapter 8 gene expression

Chapter 8Gene Expression

The Flow of Genetic Information from DNA via RNA to Protein


Outline of chapter 8

Outline of Chapter 8

  • The genetic code

    • How triplets of the four nucleotides unambiguously specify 20 amino acids, making it possible to translate information from a nucleotide chain to a sequence of amino acids

  • Transcription

    • How RNA polymerase, guided by base pairing, synthesizes a single-stranded mRNA copy of a gene’s DNA template

  • Translation

    • How base pairing between mRNA and tRNAs directs the assembly of a polypeptide on the ribosome

  • Significant differences in gene expression between prokaryotes and eukaryotes

  • How mutations affect gene information and expression


The triplet codon represents each amino acid

The 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 triplet combinations that code for amino acids


The genetic code 61 triplet codons represent 20 amino acids 3 triplet codons signify stop

The Genetic Code: 61 triplet codons represent 20 amino acids; 3 triplet codons signify stop

Fig. 8.3


A gene s nucleotide sequence is colinear the amino acid sequence of the encoded polypeptide

A gene’s nucleotide sequence is colinear the amino acid sequence of the encoded polypeptide

  • Charles Yanofsky – E. coli genes for a subunit of tyrptophan synthetase compared mutations within a gene to particular amino acid substitutions

  • Trp- mutants in trpA

  • Fine structure recombination map

  • Determined amino acid sequences of mutants


Chapter 8 gene expression

Fig. 8.4


Chapter 8 gene expression

  • A codon is composed of more than one nucleotide

    • Different point mutations may affect same amino acid

    • Codon contains more than one nucleotide

  • Each nucleotide is part of only a single codon

    • Each point mutation altered only one amino acid


Chapter 8 gene expression

A codon is composed of three nucleotides and the starting point of each gene establishes a reading framestudies of frameshift mutations in bacteriophage T4 rIIB gene

Fig. 8.5


Chapter 8 gene expression

  • Most amino acids are specified by more than one codon

  • Phenotypic effect of frameshifts depends on if reading frame is restored

Fig. 8.6


Cracking the code biochemical manipulations revealed which codons represent which amino acids

Cracking the code: biochemical manipulations revealed which codons represent which amino acids

  • The discovery of messenger RNAs, molecules for transporting genetic information

    • Protein synthesis takes place in cytoplasm deduced from radioactive tagging of amino acids

  • RNA, an intermediate molecule made in nucleus and transports DNA information to cytoplasm


Synthetic mrnas and in vitro translation determines which codons designate which amino acids

Synthetic mRNAs and in vitro translation determines which codons designate which amino acids

  • 1961 – Marshall Nirenberg and Heinrich Mathaei created mRNAs and translated to polypeptides in vitro

  • Polymononucleotides

  • Polydinucleotides

  • Polytrinucleotides

  • Polytetranucleotides

  • Read amino acid sequence and deduced codons

Fig. 8.7


Chapter 8 gene expression

  • Ambiguities resolved by Nirenberg and Philip Leder using trinucleotide mRNAs of known sequence to tRNAs charged with radioactive amino acid with ribosomes

Fig. 8.8


Chapter 8 gene expression

  • 5’ to 3’ direction of mRNA corresponds to N-terminal-to-C-terminal direction of polypeptide

    • One strand of DNA is a template

    • The other is an RNA-like strand

  • Nonsense codons cause termination of a polypeptide chain – UAA (ocher), UAG (amber), and UGA (opal)

Fig. 8.9


Summary

Summary

  • Codon consist of a triplet codon each of which specifies an amino acid

    • Code shows a 5’ to 3’ direction

  • Codons are nonoverlapping

  • Code includes three stop codons, UAA, UAG, and UGA that terminate translation

  • Code is degenerate

  • Fixed starting point establishes a reading frame

    • UAG in an initiation codon which specifies reading frame

  • 5’- 3’ direction of mRNA corresponds with N-terminus to C-terminus of polypeptide

  • Mutation modify message encoded in sequence

    • Frameshift mutaitons change reading frame

    • Missense mutations change codon of amino acid to another amino acid

    • Nonsense mutations change a codon for an amino acid to a stop codon


Do living cells construct polypeptides according to same rules as in vitro experiments

Do living cells construct polypeptides according to same rules as in vitro experiments?

  • Studies of how mutations affect amino-acid composition of polypeptides encoded by a gene

  • Missense mutations induced by mutagens should be single nucleotide substitutions and conform to the code

Fig. 8.10 a


Chapter 8 gene expression

  • 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

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 perfect correspondence between codons and amino acids among all organisms


Transcription

Transcription

  • RNA polymerase catalyzes transcription

  • Promoters signal RNA polymerase where to begin transcription

  • RNA polymerase adds nucleotides in 5’ to 3’ direction

  • Terminator sequences tell RNA when to stop transcription


Initiation of transcription

Initiation of transcription

Fig. 8.11 a


Elongation

Elongation

Fig. 8.11 b


Termination

Termination

Fig. 8.11 c


Information flow

Information flow

Fig. 8.11 d


Promoters of 10 different bacterial genes

Promoters of 10 different bacterial genes

Fig. 8.12


In eukaryotes rna is processed after transcription

In eukaryotes, RNA is processed after transcription

  • A 5’ methylated cap and a 3’ Poly-A tail are added

  • Structure of the methylated cap


How poly a tail is added to 3 end of mrna

How Poly-A tail is added to 3’ end of mRNA

Fig. 8.14


Rna splicing removes introns

RNA splicing removes introns

  • Exons – sequences found in a gene’s DNA and mature mRNA (expressed regions)

  • Introns – sequences found in DNA but not in mRNA (intervening regions)

  • Some eukaryotic genes have many introns


Dystrophin gene underlying duchenne muscular dystrophy dmd is an extreme example of introns

Dystrophin gene underlying Duchenne muscular dystrophy (DMD) is an extreme example of introns

Fig. 8.15


How rna processing splices out introns and adjoins adjacent exons

How RNA processing splices out introns and adjoins adjacent exons

Fig. 8.16


Chapter 8 gene expression

  • Splicing is catalyzed by spliceosomes

    • Ribozymes – RNA molecules that act as enzymes

    • Ensures that all splicing reactions take place in concert

Fig. 8.17


Chapter 8 gene expression

  • Alternative splicing

    • Different mRNAs can be produced by same transcript

    • Rare transplicing events combine exons from different genes

Fig. 8.18


Translation

Translation

  • Transfer RNAs (tRNAs) mediate translation of mRNA codons to amino acids

    • tRNAs carry anticodon on one end

      • Three nucleotides complementary to an mRNA codon

    • Structure of tRNA

      • Primary – nucleotide sequence

      • Secondary – short complementary sequences pair and make clover leaf shape

      • Tertiary – folding into three dimensional space shape like an L

    • Base pairing between an mRNA codon and a tRNA anticodon directs amino acid incorporation into a growing polypeptide

    • Charged tRNA is covalently coupled to its amino acid


Secondary and tertiary structure

Secondary and tertiary structure

Fig. 8.19 b


Aminoacyl trna syntetase catalyzes attachment of trnas to corresponding amino acid

Aminoacyl-tRNA syntetase catalyzes attachment of tRNAs to corresponding amino acid

Fig. 8.20


Chapter 8 gene expression

Base pairing between mRNA codon and tRNA anticodon determines where incorporation of amino acid occurs

Fig. 8.21


Wobble some trnas recognize more than one codon for amino acids they carry

Wobble: Some tRNAs recognize more than one codon for amino acids they carry

Fig. 8.22


Rhibosomes are site of polypeptide synthesis

Rhibosomes are site of polypeptide synthesis

  • Ribosomes are complex structures composed of RNA and protein

Fig. 8.23


Mechanism of translation

Mechanism of translation

  • Initiation sets stage for polypeptide synthesis

    • AUG start codon at 5’ end of mRNA

    • Formalmethionine (fMet) on initiation tRNA

      • First amino acid incorporated in bacteria

  • Elongation during which amino acids are added to growing polypeptide

    • Ribosomes move in 5’-3’ direction revealing codons

    • Addition of amino acids to C terminus

    • 2-15 amino acids per second

  • Termination which halts polypeptide synthesis

    • Nonsense codon recognized at 3’ end of reading frame

    • Release factor proteins bind at nonsense codons and halt polypeptide synthesis


Initiation of translation

Initiation of translation

Fig. 8.24 a


Elongation1

Elongation

Fig. 8.24 b


Termination of translation

Termination of translation

Fig. 8.24 c


Chapter 8 gene expression

  • Posttranslational processing can modify polypeptide structure

Fig. 8.25


Significant differences in gene expression between prokaryotes and eukaryotes

Significant differences in gene expression between prokaryotes and eukaryotes

  • Eukaryotes, nuclear membrane prevents coupling of transcription and translation

  • Prokaryotic messages are polycistronic

    • Contain information for multiple genes

  • Eukaryotes, small ribosomal subunit binds to 5’ methylated cap and migrates to AUG start codon

    • 5’ untranslated leader sequence – between 5’ cap and AUG start

    • Only a single polypeptide produced from each gene

  • Initiating tRNA in prokaryotes is fMet

  • Initiating tRNA in eukaryotes is by unmodified Met.


Chapter 8 gene expression

  • Nonsense suppression

    • (a) Nonsense mutation that causes incomplete nonfunctional polypeptide

    • (b) Nonsense-suppressing mutation causes addition of amino acid at stop codon allowing production of full length polypeptide

Fig. 8.28


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