Chapter 8 Gene Expression The Flow of Genetic

Chapter 8 Gene Expression The Flow of Genetic Information from DNA via RNA to Protein

Outline of Chapter 8 n The genetic code n n Transcription n How RNA polymerase, guided by base pairing, synthesizes a single-stranded m. RNA copy of a gene’s DNA template Translation n n 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 How base pairing between m. RNA and t. RNAs 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 n 20 amino acids encoded for by 4 nucleotides n By deduction: n 1 nucleotide/amino acid = 41 = 4 triplet combinations n 2 nucleotides/amino acid = 42 = 16 triplet combinations n 3 nucleotides/amino acid = 43 = 64 triplet combinations n 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 Fig. 8. 3

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 n Trp- mutants in trp. A n Fine structure recombination map n Determined amino acid sequences of mutants n

Fig. 8. 4

n A codon is composed of more than one nucleotide Different point mutations may affect same amino acid n Codon contains more than one nucleotide n n Each nucleotide is part of only a single codon n Each point mutation altered only one amino acid

A codon is composed of three nucleotides and the starting point of each gene establishes a reading frame studies of frameshift mutations in bacteriophage T 4 r. IIB gene Fig. 8. 5

n n Fig. 8. 6 Most amino acids are specified by more than one codon Phenotypic effect of frameshifts depends on if reading frame is restored

Cracking the code: biochemical manipulations revealed which codons represent which amino acids n The discovery of messenger RNAs, molecules for transporting genetic information n n 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 m. RNAs and in vitro translation determines which codons designate which amino acids n n n 1961 – Marshall Nirenberg and Heinrich Mathaei created m. RNAs and translated to polypeptides in vitro Polymononucleotides Polydinucleotides Polytrinucleotides Polytetranucleotides Read amino acid sequence and deduced codons Fig. 8. 7

n Fig. 8. 8 Ambiguities resolved by Nirenberg and Philip Leder using trinucleotide m. RNAs of known sequence to t. RNAs charged with radioactive amino acid with ribosomes

n 5’ to 3’ direction of m. RNA corresponds to N-terminal-to-C -terminal direction of polypeptide n n n 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 n Codon consist of a triplet codon each of which specifies an amino acid n n n 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 n n n Code shows a 5’ to 3’ direction UAG in an initiation codon which specifies reading frame 5’- 3’ direction of m. RNA corresponds with N-terminus to Cterminus of polypeptide Mutation modify message encoded in sequence n n n 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? n n Fig. 8. 10 a 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

Proflavin treatment generates Trp- mutants n Further treatment generates Trp+ revertants n n Single base insertion (Trp-) and a deletion causes reversion (Trp+) Fig. 8. 10 b

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

Transcription RNA polymerase catalyzes transcription n Promoters signal RNA polymerase where to begin transcription n RNA polymerase adds nucleotides in 5’ to 3’ direction n Terminator sequences tell RNA when to stop transcription n

Initiation of transcription Fig. 8. 11 a

Elongation Fig. 8. 11 b

Termination Fig. 8. 11 c

Information flow Fig. 8. 11 d

Promoters of 10 different bacterial genes Fig. 8. 12

In eukaryotes, RNA is processed after transcription n n 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 m. RNA Fig. 8. 14

RNA splicing removes introns Exons – sequences found in a gene’s DNA and mature m. RNA (expressed regions) n Introns – sequences found in DNA but not in m. RNA (intervening regions) n Some eukaryotic genes have many introns n

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 Fig. 8. 16

n Splicing is catalyzed by spliceosomes Ribozymes – RNA molecules that act as enzymes n Ensures that all splicing reactions take place in concert n Fig. 8. 17

n Alternative splicing Different m. RNAs can be produced by same transcript n Rare transplicing events combine exons from different genes n Fig. 8. 18

Translation n Transfer RNAs (t. RNAs) mediate translation of m. RNA codons to amino acids n t. RNAs carry anticodon on one end n n Structure of t. RNA n n n Three nucleotides complementary to an m. RNA codon 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 m. RNA codon and a t. RNA anticodon directs amino acid incorporation into a growing polypeptide Charged t. RNA is covalently coupled to its amino acid

Secondary and tertiary structure Fig. 8. 19 b

Aminoacyl-t. RNA syntetase catalyzes attachment of t. RNAs to corresponding amino acid Fig. 8. 20

Base pairing between m. RNA codon and t. RNA anticodon determines where incorporation of amino acid occurs Fig. 8. 21

Wobble: Some t. RNAs recognize more than one codon for amino acids they carry Fig. 8. 22

Rhibosomes are site of polypeptide synthesis n Ribosomes are complex structures composed of RNA and protein Fig. 8. 23

Mechanism of translation n Initiation sets stage for polypeptide synthesis n n AUG start codon at 5’ end of m. RNA Formalmethionine (f. Met) on initiation t. RNA n n Elongation during which amino acids are added to growing polypeptide n n First amino acid incorporated in bacteria 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 n n Nonsense codon recognized at 3’ end of reading frame Release factor proteins bind at nonsense codons and halt polypeptide synthesis

Initiation of translation Fig. 8. 24 a

Elongation Fig. 8. 24 b

Termination of translation Fig. 8. 24 c

n Posttranslation al processing can modify polypeptide structure Fig. 8. 25

Significant differences in gene expression between prokaryotes and eukaryotes n n Eukaryotes, nuclear membrane prevents coupling of transcription and translation Prokaryotic messages are polycistronic n n Eukaryotes, small ribosomal subunit binds to 5’ methylated cap and migrates to AUG start codon n n Contain information for multiple genes 5’ untranslated leader sequence – between 5’ cap and AUG start Only a single polypeptide produced from each gene Initiating t. RNA in prokaryotes is f. Met Initiating t. RNA in eukaryotes is by unmodified Met.

n Fig. 8. 28 Nonsense suppression n (a) Nonsense mutation that causes incomplete nonfunctional polypeptide n (b) Nonsensesuppressing mutation causes addition of amino acid at stop codon allowing production of full length polypeptide