Chapter 8 Gene Expression

Chapter 8Chapter 8

Gene ExpressionGene ExpressionThe Flow of Genetic Information The Flow of Genetic Information from DNA via RNA to Proteinfrom DNA via RNA to Protein

Outline of Chapter 8Outline of Chapter 8 The genetic codeThe genetic code

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

TranscriptionTranscription How RNA polymerase, guided by base pairing, synthesizes a How RNA polymerase, guided by base pairing, synthesizes a

single-stranded mRNA copy of a gene’s DNA templatesingle-stranded mRNA copy of a gene’s DNA template TranslationTranslation

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

Significant differences in gene expression between Significant differences in gene expression between prokaryotes and eukaryotesprokaryotes and eukaryotes

How mutations affect gene information and expressionHow mutations affect gene information and expression

The triplet codon represents each The triplet codon represents each amino acidamino acid

20 amino acids encoded for by 4 nucleotides20 amino acids encoded for by 4 nucleotides By deduction: By deduction:

1 nucleotide/amino acid = 41 nucleotide/amino acid = 411 = 4 triplet combinations = 4 triplet combinations 2 nucleotides/amino acid = 42 nucleotides/amino acid = 422 = 16 triplet = 16 triplet

combinationscombinations 3 nucleotides/amino acid = 43 nucleotides/amino acid = 433 = 64 triplet = 64 triplet

combinationscombinations Must be at least triplet combinations that code Must be at least triplet combinations that code

for amino acidsfor amino acids

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

Fig. 8.3

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

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

TrpTrp-- mutants in trpA mutants in trpA Fine structure recombination mapFine structure recombination map Determined amino acid sequences of Determined amino acid sequences of

mutantsmutants

Fig. 8.4

A codon is composed of more than one A codon is composed of more than one nucleotidenucleotide Different point mutations may affect same Different point mutations may affect same

amino acidamino acid Codon contains more than one nucleotideCodon contains more than one nucleotide

Each nucleotide is part of only a single Each nucleotide is part of only a single codoncodon Each point mutation altered only one amino Each point mutation altered only one amino

acidacid

A codon is composed of three nucleotides and the starting A codon is composed of three nucleotides and the starting point of each gene establishes a reading framepoint of each gene establishes a reading frame

studies of frameshift mutations in bacteriophage T4 rIIB genestudies of frameshift mutations in bacteriophage T4 rIIB gene

Fig. 8.5

Most amino acids Most amino acids are specified by are specified by more than one more than one codoncodon

Phenotypic effect Phenotypic effect of frameshifts of frameshifts depends on if depends on if reading frame is reading frame is restoredrestored

Fig. 8.6

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

The discovery of messenger RNAs, The discovery of messenger RNAs, molecules for transporting genetic molecules for transporting genetic informationinformation Protein synthesis takes place in cytoplasm Protein synthesis takes place in cytoplasm

deduced from radioactive tagging of amino deduced from radioactive tagging of amino acidsacids

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

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

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

PolymononucleotidesPolymononucleotides PolydinucleotidesPolydinucleotides PolytrinucleotidesPolytrinucleotides PolytetranucleotidesPolytetranucleotides Read amino acid sequence Read amino acid sequence

and deduced codonsand deduced codons

Fig. 8.7

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

Fig. 8.8

5’ to 3’ direction of mRNA corresponds to N-terminal-to-5’ to 3’ direction of mRNA corresponds to N-terminal-to-C-terminal direction of polypeptideC-terminal direction of polypeptide One strand of DNA is a templateOne strand of DNA is a template The other is an RNA-like strandThe other is an RNA-like strand

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

Fig. 8.9

SummarySummary Codon consist of a triplet codon each of which specifies an amino Codon consist of a triplet codon each of which specifies an amino

acidacid Code shows a 5’ to 3’ directionCode shows a 5’ to 3’ direction

Codons are nonoverlappingCodons are nonoverlapping Code includes three stop codons, UAA, UAG, and UGA that Code includes three stop codons, UAA, UAG, and UGA that

terminate translationterminate translation Code is degenerateCode is degenerate Fixed starting point establishes a reading frameFixed starting point establishes a reading frame

UAG in an initiation codon which specifies reading frameUAG in an initiation codon which specifies reading frame 5’- 3’ direction of mRNA corresponds with N-terminus to C-5’- 3’ direction of mRNA corresponds with N-terminus to C-

terminus of polypeptideterminus of polypeptide Mutation modify message encoded in sequenceMutation modify message encoded in sequence

Frameshift mutaitons change reading frameFrameshift mutaitons change reading frame Missense mutations change codon of amino acid to another amino acidMissense mutations change codon of amino acid to another amino acid Nonsense mutations change a codon for an amino acid to a stop codonNonsense mutations change a codon for an amino acid to a stop codon

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

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

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

Fig. 8.10 a

Proflavin treatment generates TrpProflavin treatment generates Trp-- mutants mutants Further treatment generates TrpFurther treatment generates Trp++

revertantsrevertants Single base insertion (TrpSingle base insertion (Trp--) and a deletion ) and a deletion

causes reversion (Trpcauses reversion (Trp++))

Fig. 8.10 b

Genetic code is almost universal but Genetic code is almost universal but not quitenot quite

All living organisms use same basic genetic All living organisms use same basic genetic codecode Translational systems can use mRNA from Translational systems can use mRNA from

another organism to generate proteinanother organism to generate protein Comparisons of DNA and protein sequence Comparisons of DNA and protein sequence

reveal perfect correspondence between codons reveal perfect correspondence between codons and amino acids among all organismsand amino acids among all organisms

TranscriptionTranscription

RNA polymerase catalyzes transcriptionRNA polymerase catalyzes transcription Promoters signal RNA polymerase where to Promoters signal RNA polymerase where to

begin transcriptionbegin transcription RNA polymerase adds nucleotides in 5’ to 3’ RNA polymerase adds nucleotides in 5’ to 3’

directiondirection Terminator sequences tell RNA when to Terminator sequences tell RNA when to

stop transcriptionstop transcription

Initiation of transcriptionInitiation of transcription

Fig. 8.11 a

ElongationElongation

Fig. 8.11 b

TerminationTermination

Fig. 8.11 c

Information flowInformation flow

Fig. 8.11 d

Promoters of 10 different bacterial genesPromoters of 10 different bacterial genes

Fig. 8.12

In eukaryotes, RNA is processed In eukaryotes, RNA is processed after transcriptionafter 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 mRNAHow Poly-A tail is added to 3’ end of mRNA

Fig. 8.14

RNA splicing removes intronsRNA splicing removes introns

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

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

Some eukaryotic genes have many intronsSome eukaryotic genes have many introns

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

Fig. 8.15

How RNA processing splices out How RNA processing splices out introns and adjoins adjacent exonsintrons and adjoins adjacent exons

Fig. 8.16

Splicing is Splicing is catalyzed by catalyzed by spliceosomesspliceosomes Ribozymes – Ribozymes –

RNA molecules RNA molecules that act as that act as enzymesenzymes

Ensures that all Ensures that all splicing reactions splicing reactions take place in take place in concertconcert

Fig. 8.17

Alternative Alternative splicingsplicing Different mRNAs Different mRNAs

can be produced can be produced by same by same transcripttranscript

Rare transplicing Rare transplicing events combine events combine exons from exons from different genesdifferent genes

Fig. 8.18

TranslationTranslation Transfer RNAs (tRNAs) mediate translation of Transfer RNAs (tRNAs) mediate translation of

mRNA codons to amino acidsmRNA codons to amino acids tRNAs carry anticodon on one endtRNAs carry anticodon on one end

Three nucleotides complementary to an mRNA codonThree nucleotides complementary to an mRNA codon Structure of tRNAStructure of tRNA

Primary – nucleotide sequencePrimary – nucleotide sequence Secondary – short complementary sequences pair and make Secondary – short complementary sequences pair and make

clover leaf shapeclover leaf shape Tertiary – folding into three dimensional space shape like an LTertiary – folding into three dimensional space shape like an L

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

Charged tRNA is covalently coupled to its amino acidCharged tRNA is covalently coupled to its amino acid

Secondary and tertiary structureSecondary and tertiary structure

Fig. 8.19 b

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

Fig. 8.20

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

Fig. 8.21

Wobble: Wobble: Some tRNAs Some tRNAs

recognize recognize more than more than

one codon for one codon for amino acids amino acids they carrythey carry

Fig. 8.22

Rhibosomes are site of polypeptide synthesisRhibosomes are site of polypeptide synthesis

Ribosomes Ribosomes are complex are complex structures structures composed of composed of RNA and RNA and proteinprotein

Fig. 8.23

Mechanism of translationMechanism of translation

Initiation sets stage for polypeptide synthesisInitiation sets stage for polypeptide synthesis AUG start codon at 5’ end of mRNAAUG start codon at 5’ end of mRNA Formalmethionine (fMet) on initiation tRNA Formalmethionine (fMet) on initiation tRNA

First amino acid incorporated in bacteriaFirst amino acid incorporated in bacteria

Elongation during which amino acids are added to Elongation during which amino acids are added to growing polypeptidegrowing polypeptide Ribosomes move in 5’-3’ direction revealing codonsRibosomes move in 5’-3’ direction revealing codons Addition of amino acids to C terminusAddition of amino acids to C terminus 2-15 amino acids per second2-15 amino acids per second

Termination which halts polypeptide synthesisTermination which halts polypeptide synthesis Nonsense codon recognized at 3’ end of reading frameNonsense codon recognized at 3’ end of reading frame Release factor proteins bind at nonsense codons and halt Release factor proteins bind at nonsense codons and halt

polypeptide synthesispolypeptide synthesis

Initiation of translationInitiation of translation

Fig. 8.24 a

ElongationElongation

Fig. 8.24 b

Termination of translationTermination of translation

Fig. 8.24 c

PosttranslationPosttranslational processing al processing can modify can modify polypeptide polypeptide structurestructure

Fig. 8.25

Significant differences in gene expression Significant differences in gene expression between prokaryotes and eukaryotesbetween prokaryotes and eukaryotes

Eukaryotes, nuclear membrane prevents coupling of Eukaryotes, nuclear membrane prevents coupling of transcription and translationtranscription and translation

Prokaryotic messages are polycistronicProkaryotic messages are polycistronic Contain information for multiple genesContain information for multiple genes

Eukaryotes, small ribosomal subunit binds to 5’ Eukaryotes, small ribosomal subunit binds to 5’ methylated cap and migrates to AUG start codonmethylated cap and migrates to AUG start codon 5’ untranslated leader sequence – between 5’ cap and AUG start5’ untranslated leader sequence – between 5’ cap and AUG start Only a single polypeptide produced from each geneOnly a single polypeptide produced from each gene

Initiating tRNA in prokaryotes is fMetInitiating tRNA in prokaryotes is fMet Initiating tRNA in eukaryotes is by unmodified Met.Initiating tRNA in eukaryotes is by unmodified Met.

Nonsense Nonsense suppressionsuppression (a) Nonsense (a) Nonsense

mutation that mutation that causes incomplete causes incomplete nonfunctional nonfunctional polypeptidepolypeptide

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

Fig. 8.28