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: 1 nucleotide/amino acid = 4 1 = 4 triplet combinations 1 nucleotide/amino acid = 4 1 = 4 triplet combinations 2 nucleotides/amino acid = 4 2 = 16 triplet combinations 2 nucleotides/amino acid = 4 2 = 16 triplet combinations 3 nucleotides/amino acid = 4 3 = 64 triplet combinations 3 nucleotides/amino acid = 4 3 = 64 triplet combinations Must be at least 64 triplet combinations that code for 20 amino acids Must be at least 64 triplet combinations that code for 20 amino acids

The Genetic Code: 61 triplet codons represent 20 amino acids; 3 triplet codons signify stop Fig. 8.3

The nucleotide sequence of a gene is colinear with the amino acid sequence of the polypeptide Charles Yanofsky – compared mutations within a gene to particular amino acid substitutions Charles Yanofsky – compared mutations within a gene to particular amino acid substitutions Trp - mutants in the trpA gene that encodes tryptophan synthetase Trp - mutants in the trpA gene that encodes tryptophan synthetase Fine structure recombination map Fine structure recombination map Determined amino acid sequences of mutants Determined amino acid sequences of mutants

Fig. 8.4

A codon is composed of more than one nucleotide A codon is composed of more than one nucleotide Different point mutations may affect same amino acid Different point mutations may affect same amino acid Each nucleotide is part of only a single codon Each nucleotide is part of only a single codon Each point mutation altered only one amino acid Each point mutation altered only one amino acid Yanofsky’s conclusions

A codon is composed of three nucleotides and the starting point of each gene establishes a reading frame studies of frameshift mutations in bacteriophage T4 rIIB gene Fig. 8.5

Most amino acids are specified by more than one codon Most amino acids are specified by more than one codon Phenotypic effect of frameshifts depends on reading frame Phenotypic effect of frameshifts depends on reading frame Fig. 8.6

Cracking the code: biochemical manipulations revealed which codons represent which amino acids The discovery of messenger RNAs The discovery of messenger RNAs Protein synthesis takes place in cytoplasm deduced from radioactive tagging of amino acids Protein synthesis takes place in cytoplasm deduced from radioactive tagging of amino acids An intermediate molecule made in nucleus DNA information to cytoplasm An intermediate molecule made in nucleus DNA information to cytoplasm

Synthetic mRNAs and in vitro translation determines which codons designate which amino acids 1961 – Marshall Nirenberg and Heinrich Mathaei created mRNAs and translated in vitro 1961 – Marshall Nirenberg and Heinrich Mathaei created mRNAs and translated in vitro Polymononucleotides Polymononucleotides Polydinucleotides Polydinucleotides Polytrinucleotides Polytrinucleotides Polytetranucleotides Polytetranucleotides Determined amino acid sequence to deduce codons Determined amino acid sequence to deduce codons Fig. 8.7

Ambiguities resolved by Nirenberg and Philip Leder using trinucleotide mRNAs of known sequence and tRNAs charged with a radioactive amino acid Ambiguities resolved by Nirenberg and Philip Leder using trinucleotide mRNAs of known sequence and tRNAs charged with a radioactive amino acid Fig. 8.8

5’ to 3’ direction of mRNA corresponds to N-terminal-to- C-terminal direction of polypeptide 5’ to 3’ direction of mRNA corresponds to N-terminal-to- C-terminal direction of polypeptide Nonsense codons cause termination of a polypeptide chain – UAA (ochre), UAG (amber), and UGA (opal) Nonsense codons cause termination of a polypeptide chain – UAA (ochre), UAG (amber), and UGA (opal) Fig. 8.9

Do living cells construct polypeptides according to same rules as in vitro experiments? How gene mutations affect amino-acid composition How gene mutations affect amino-acid composition Missense mutations should conform to the code Missense mutations should conform to the code Fig a

Proflavin treatment generates trp - mutants Proflavin treatment generates trp - mutants Further treatment generates trp + revertants Further treatment generates trp + revertants Single base insertion (trp - ) and a deletion causes reversion (trp + ) Single base insertion (trp - ) and a deletion causes reversion (trp + ) Fig b

Genetic code is almost universal but not quite All living organisms use same basic genetic code All living organisms use same basic genetic code Translational systems can use mRNA from another organism to generate protein Translational systems can use mRNA from another organism to generate protein Comparisons of DNA and protein sequence reveal correspondence between codons and amino acids among all organisms Comparisons of DNA and protein sequence reveal correspondence between codons and amino acids among all organisms

Specialized example of regulation through RNA stability Fig

Promoters of 10 different bacterial genes Fig. 8.12

Regulatory elements that map near a gene are cis-acting DNA sequences cis-acting elements cis-acting elements Promoter – very close to initiation site Promoter – very close to initiation site Enhancer Enhancer Can be far way from gene Can be far way from gene Can be in either orientation Can be in either orientation Function to augment or repress basal levels of transcription Function to augment or repress basal levels of transcription Fig a

Fig. 16.2

In eukaryotes three RNA polymerases transcribe different sets of genes RNA polymerase I transcribes rRNA RNA polymerase I transcribes rRNA rRNAs are made of tandem repeats on one or more chromosomes rRNAs are made of tandem repeats on one or more chromosomes RNA polymerase I transcribes one primary transcript which is broken down into 28s and 5.8s by processing RNA polymerase I transcribes one primary transcript which is broken down into 28s and 5.8s by processing Fig a

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

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

Reporter constructs are a tool for studying gene regulation Sequence of DNA containing regulatory region, but not coding region Sequence of DNA containing regulatory region, but not coding region Coding region replaced with easily identifiable product Coding region replaced with easily identifiable product In vitro mutagenesis can be used to systematically alter the presumptive regulatory region In vitro mutagenesis can be used to systematically alter the presumptive regulatory region

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

Creating a collection of lacZ insertions in the chromosome Fig b

Use of a fusion to overproduce a gene product Fig c

Fig. C.8 Reporter constructs in worms

GFP tagging can be used to follow the localization of proteins Recombinant gene encoding a GFP fusion protein at C terminus Recombinant gene encoding a GFP fusion protein at C terminus Mouse with GFP- labeled transgene expressed throughout body Mouse with GFP- labeled transgene expressed throughout body Fig c,d

Enhancer trapping to identify genes by expression pattern P element with lacZ gene downstream of promoter P element with lacZ gene downstream of promoter When mobilized, 65% of new insertions express lacZ reporter during development When mobilized, 65% of new insertions express lacZ reporter during development Promoter can only activate transcription if under control of enhancers of genes near insertion site Promoter can only activate transcription if under control of enhancers of genes near insertion site Detects genes turned on in certain tissues Detects genes turned on in certain tissues Genes isolated by plasmid rescue Genes isolated by plasmid rescue Fig. D.10

Regulatory elements that map far from a gene are trans-acting DNA sequences Proteins that interact directly or indirectly with cis- acting elements Proteins that interact directly or indirectly with cis- acting elements Transcription factors Transcription factors Identified by: Identified by: Biochemical studies to identify proteins that bind in vitro to cis-acting elements Biochemical studies to identify proteins that bind in vitro to cis-acting elements Fig b

trans-acting proteins control transcription from class II promoters Basal factors bind to the promoter Basal factors bind to the promoter TBP – TATA box binding protein TBP – TATA box binding protein TAF – TBP associated factors TAF – TBP associated factors RNA polymerase II binds to basal factors RNA polymerase II binds to basal factors Fig a

Most regulatory proteins are oligomeric  More than one binding domain  DNase footprint identifies binding region  DNase cannot digest protein covered sites Fig a

Activating factors Bind to enhancer DNA in specific ways Bind to enhancer DNA in specific ways Interact with other proteins to activate and increase transcription as much as 100-fold above basal levels Interact with other proteins to activate and increase transcription as much as 100-fold above basal levels Two structural domains mediate these functions Two structural domains mediate these functions DNA-binding domain DNA-binding domain Transcription-activator domain Transcription-activator domain

Transcriptional activators bind to specific enhancers at specific times to increase transcriptional levels Transcriptional activators bind to specific enhancers at specific times to increase transcriptional levels Fig a

helix-loop- helix and zinc-finger proteins bind to the DNA binding domains of enhancer elements helix-loop- helix and zinc-finger proteins bind to the DNA binding domains of enhancer elements Examples of common transcription factors Fig b

Leucine zipper – a common activator protein with dimerization domains Fig b

Some eukaryotic activators must form dimers to function Eukaryotic transcription factor protein structure Eukaryotic transcription factor protein structure Homomers – multimeric proteins composed of identical subunits Homomers – multimeric proteins composed of identical subunits Heteromers – multimeric proteins composed of nonidentical subunits Heteromers – multimeric proteins composed of nonidentical subunits Fig a

Localization of activator domains using recombinant DNA constructs Fusion constructs from three parts of gene encoding an activator protein Fusion constructs from three parts of gene encoding an activator protein Reporter gene can only be transcribed if activator domain is present in the fusion construct Reporter gene can only be transcribed if activator domain is present in the fusion construct Part B contains activation domain, but not part A or C Part B contains activation domain, but not part A or C Fig. 17.6