A triplet codon represents each amino acid 20 amino acids encoded for by 4 nucleotides 20 amino acids encoded for by 4 nucleotides By deduction: By deduction:

A triplet codon represents each A 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 64 triplet combinations that Must be at least 64 triplet combinations that

code for 20 amino acidscode for 20 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

The nucleotide sequence of a gene is colinear with The nucleotide sequence of a gene is colinear with the amino acid sequence of the polypeptidethe amino acid sequence of the polypeptide

Charles Yanofsky – compared mutations Charles Yanofsky – compared mutations within a gene to particular amino acid within a gene to particular amino acid substitutionssubstitutions

TrpTrp-- mutants in the trpA gene that encodes mutants in the trpA gene that encodes tryptophan synthetasetryptophan synthetase

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 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

Yanofsky’s conclusionsYanofsky’s conclusions

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 depends on reading framereading frame

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 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

An intermediate molecule made in nucleus DNA An intermediate molecule made in nucleus DNA information to cytoplasminformation to cytoplasm

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 in vitrotranslated in vitro

PolymononucleotidesPolymononucleotides PolydinucleotidesPolydinucleotides PolytrinucleotidesPolytrinucleotides PolytetranucleotidesPolytetranucleotides Determined amino acid Determined amino acid

sequence to deduce codonssequence to deduce 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 and sequence and tRNAs charged tRNAs charged with a radioactive with a radioactive amino acidamino acid

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

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

Fig. 8.9

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?

How gene mutations How gene mutations affect amino-acid affect amino-acid composition composition

Missense mutations Missense mutations should conform to should conform to the codethe code

Fig. 8.10 a

Proflavin treatment generates trpProflavin treatment generates trp-- mutants mutants Further treatment generates trpFurther treatment generates trp++ revertants revertants

Single base insertion (trpSingle base insertion (trp--) and a deletion causes ) and a deletion causes reversion (trpreversion (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 correspondence between codons and reveal correspondence between codons and amino acids among all organismsamino acids among all organisms

Specialized Specialized example of example of regulation regulation through through

RNA RNA stabilitystability

Fig. 17.17

Promoters of 10 different bacterial genesPromoters of 10 different bacterial genes

Fig. 8.12

Regulatory elements that map near a Regulatory elements that map near a gene are gene are ciscis-acting DNA sequences-acting DNA sequences

ciscis-acting elements-acting elements Promoter – very close to initiation sitePromoter – very close to initiation site Enhancer Enhancer

Can be far way from geneCan be far way from gene Can be in either orientationCan be in either orientation Function to augment or repress basal levels of transcriptionFunction to augment or repress basal levels of transcription

Fig. 17.1 a

Fig. 16.2

In eukaryotes three RNA polymerases In eukaryotes three RNA polymerases transcribe different sets of genestranscribe different sets of genes

RNA polymerase I RNA polymerase I transcribes rRNAtranscribes rRNA rRNAs are made of rRNAs are made of

tandem repeats on tandem repeats on one or more one or more chromosomeschromosomes

RNA polymerase I RNA polymerase I transcribes one transcribes one primary transcript primary transcript which is broken which is broken down into 28s and down into 28s and 5.8s by processing5.8s by processing

Fig. 17.2 a

RNA polymerase III transcribes RNA polymerase III transcribes tRNAs and other small RNAs (5s tRNAs and other small RNAs (5s rRNA, snRNAs)rRNA, snRNAs)

Fig. 17.2 b

RNA polymerase II transcribes all protein RNA polymerase II transcribes all protein coding genescoding genes

Reporter constructs are a tool for Reporter constructs are a tool for studying gene regulationstudying gene regulation

Sequence of DNA containing regulatory Sequence of DNA containing regulatory region, but not coding regionregion, but not coding region

Coding region replaced with easily Coding region replaced with easily identifiable productidentifiable product

In vitroIn vitro mutagenesis can be used to mutagenesis can be used to systematically alter the presumptive systematically alter the presumptive regulatory regionregulatory region

Fusion used to perform genetic studies of the Fusion used to perform genetic studies of the regulatory region of gene Xregulatory region of gene X

Fig. 16.18 a

Creating a Creating a collection of collection of

lacZ lacZ insertions in insertions in

the the chromosomechromosome

Fig. 16.18 b

Use of a fusion to Use of a fusion to overproduce a gene overproduce a gene

productproduct

Fig. 16.18 c

Fig. C.8

Reporter constructs in wormsReporter constructs in worms

GFP tagging can be used to follow the GFP tagging can be used to follow the localization of proteinslocalization of proteins

Recombinant gene Recombinant gene encoding a GFP encoding a GFP fusion protein at C fusion protein at C terminusterminus

Mouse with GFP-Mouse with GFP-labeled transgene labeled transgene expressed expressed throughout bodythroughout body

Fig. 19.18 c,d

Enhancer trapping to identify genes Enhancer trapping to identify genes by expression patternby expression pattern

P element with P element with lacZlacZ gene gene downstream of promoterdownstream of promoter

When mobilized, 65% of When mobilized, 65% of new insertions express new insertions express lacZlacZ reporter during reporter during developmentdevelopment

Promoter can only activate Promoter can only activate transcription if under transcription if under control of enhancers of control of enhancers of genes near insertion sitegenes near insertion site

Detects genes turned on in Detects genes turned on in certain tissuescertain tissues

Genes isolated by plasmid Genes isolated by plasmid rescuerescue

Fig. D.10

Regulatory elements that map far from a Regulatory elements that map far from a gene are gene are transtrans-acting DNA sequences-acting DNA sequences

Proteins that Proteins that interact directly or interact directly or indirectly with indirectly with ciscis--acting elementsacting elements Transcription Transcription

factorsfactors Identified by:Identified by:

Biochemical studies Biochemical studies to identify proteins to identify proteins that bind in vitro to that bind in vitro to ciscis-acting elements-acting elements

Fig. 17.1 b

transtrans-acting proteins control transcription -acting proteins control transcription from class II promotersfrom class II promoters

Basal factors bind to Basal factors bind to the promoterthe promoter TBP – TATA box TBP – TATA box

binding proteinbinding protein TAF – TBP TAF – TBP

associated factorsassociated factors RNA polymerase II RNA polymerase II

binds to basal binds to basal factors factors

Fig. 17.4 a

Most regulatory proteins are Most regulatory proteins are oligomericoligomeric

More than one binding domain

DNase footprint identifies binding region

DNase cannot digest protein covered sites

Fig. 16.15 a

Activating factorsActivating factors

Bind to enhancer DNA in specific waysBind to enhancer DNA in specific ways Interact with other proteins to activate and Interact with other proteins to activate and

increase transcription as much as 100-fold increase transcription as much as 100-fold above basal levelsabove basal levels

Two structural domains mediate these Two structural domains mediate these functionsfunctions DNA-binding domainDNA-binding domain Transcription-activator domainTranscription-activator domain

Transcriptional Transcriptional activators bind activators bind to specific to specific enhancers at enhancers at specific times to specific times to increase increase transcriptional transcriptional levelslevels

Fig. 17.5 a

helix-loop-helix-loop-helix and helix and zinc-finger zinc-finger proteins bind proteins bind to the DNA to the DNA binding binding domains of domains of enhancer enhancer elementselements

Examples of common transcription factorsExamples of common transcription factors

Fig. 17.5 b

Leucine zipper – a common activator protein Leucine zipper – a common activator protein with dimerization domainswith dimerization domains

Fig. 17.7 b

Some eukaryotic activators must Some eukaryotic activators must form dimers to functionform dimers to function

Eukaryotic transcription factor protein structureEukaryotic transcription factor protein structure Homomers – multimeric proteins composed of identical Homomers – multimeric proteins composed of identical

subunitssubunits Heteromers – multimeric proteins composed of Heteromers – multimeric proteins composed of

nonidentical subunitsnonidentical subunits

Fig. 17.7 a

Localization of activator domains Localization of activator domains using recombinant DNA constructsusing recombinant DNA constructs

Fusion constructs Fusion constructs from three parts of from three parts of gene encoding an gene encoding an activator proteinactivator protein

Reporter gene can Reporter gene can only be transcribed only be transcribed if activator domain if activator domain is present in the is present in the fusion constructfusion construct

Part B contains Part B contains activation domain, activation domain, but not part A or Cbut not part A or C

Fig. 17.6