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

Outline of Chapter 8 The genetic code Transcription Translation 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 Transcription How RNA polymerase, guided by base pairing, synthesizes a single-stranded mRNA copy of a gene’s DNA template Translation How base pairing between mRNA and tRNAs 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 20 amino acids encoded for by 4 nucleotides By deduction: 1 nucleotide/amino acid = 41 = 4 triplet combinations 2 nucleotides/amino acid = 42 = 16 triplet combinations 3 nucleotides/amino acid = 43 = 64 triplet combinations 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 Figure 8.3 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 Trp- mutants in trpA Fine structure recombination map Determined amino acid sequences of mutants

Figure 8.4 Fig. 8.4

A codon is composed of more than one nucleotide Different point mutations may affect same amino acid Codon contains more than one nucleotide Each nucleotide is part of only a single codon 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 T4 rIIB gene Figure 8.5 Fig. 8.5

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

Cracking the code: biochemical manipulations revealed which codons represent which amino acids The discovery of messenger RNAs, molecules for transporting genetic information 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

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

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

5’ to 3’ direction of mRNA corresponds to N-terminal-to-C-terminal direction of polypeptide 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) Figure 8.9 Fig. 8.9

Summary Codon consist of a triplet codon each of which specifies an amino acid Code shows a 5’ to 3’ direction 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 UAG in an initiation codon which specifies reading frame 5’- 3’ direction of mRNA corresponds with N-terminus to C-terminus of polypeptide Mutation modify message encoded in sequence 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? 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 Figure 8.10 a Fig. 8.10 a

Proflavin treatment generates Trp- mutants Further treatment generates Trp+ revertants Single base insertion (Trp-) and a deletion causes reversion (Trp+) Figure 8.10 b Fig. 8.10 b

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

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

Initiation of transcription Figure 8.11 a Fig. 8.11 a

Elongation Figure 8.11 b Fig. 8.11 b

Termination Figure 8.11 c Fig. 8.11 c

Information flow Figure 8.11 d Fig. 8.11 d

Promoters of 10 different bacterial genes Figure 8.12 Fig. 8.12

In eukaryotes, RNA is processed after transcription Figure 8.13 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 mRNA Figure 8.14 Fig. 8.14

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

Dystrophin gene underlying Duchenne muscular dystrophy (DMD) is an extreme example of introns Figure 8.15 Fig. 8.15

How RNA processing splices out introns and adjoins adjacent exons Figure 8.16 Fig. 8.16

Splicing is catalyzed by spliceosomes Ribozymes – RNA molecules that act as enzymes Ensures that all splicing reactions take place in concert Figure 8.17 Fig. 8.17

Alternative splicing Different mRNAs can be produced by same transcript Rare transplicing events combine exons from different genes Figure 8.18 Fig. 8.18

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

Secondary and tertiary structure Figure 8.19 b Fig. 8.19 b

Aminoacyl-tRNA syntetase catalyzes attachment of tRNAs to corresponding amino acid Figure 8.20 Fig. 8.20

Base pairing between mRNA codon and tRNA anticodon determines where incorporation of amino acid occurs Figure 8.21 Fig. 8.21

Wobble: Some tRNAs recognize more than one codon for amino acids they carry Figure 8.22 Fig. 8.22

Rhibosomes are site of polypeptide synthesis Ribosomes are complex structures composed of RNA and protein Figure 8.23 Fig. 8.23

Mechanism of translation Initiation sets stage for polypeptide synthesis AUG start codon at 5’ end of mRNA Formalmethionine (fMet) on initiation tRNA First amino acid incorporated in bacteria Elongation during which amino acids are added to growing polypeptide 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 Nonsense codon recognized at 3’ end of reading frame Release factor proteins bind at nonsense codons and halt polypeptide synthesis

Initiation of translation Figure 8.24 a Fig. 8.24 a

Elongation Figure 8.24 b Fig. 8.24 b

Termination of translation Figure 8.24 c Fig. 8.24 c

Posttranslational processing can modify polypeptide structure Figure 8.25 Fig. 8.25

Significant differences in gene expression between prokaryotes and eukaryotes Eukaryotes, nuclear membrane prevents coupling of transcription and translation Prokaryotic messages are polycistronic Contain information for multiple genes Eukaryotes, small ribosomal subunit binds to 5’ methylated cap and migrates to AUG start codon 5’ untranslated leader sequence – between 5’ cap and AUG start Only a single polypeptide produced from each gene Initiating tRNA in prokaryotes is fMet Initiating tRNA in eukaryotes is by unmodified Met.

Table 8.1

Table 8 Table 8.2

Nonsense suppression (a) Nonsense mutation that causes incomplete nonfunctional polypeptide (b) Nonsense-suppressing mutation causes addition of amino acid at stop codon allowing production of full length polypeptide Figure 8.28 Fig. 8.28