Post on 23-Dec-2015
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