13-13 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

The code is nearly universal Special codons: AUG (which specifies methionine) = start codon AUG specifies additional methionines within the coding sequence UAA, UAG and UGA = termination, or stop, codons The code is degenerate More than one codon can specify the same amino acid For example: GGU, GGC, GGA and GGG all code for lysine In most instances, the third base is the degenerate base It is sometime referred to as the wobble base The code is nearly universal Only a few rare exceptions have been noted Refer to Table 13.3 13-14 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Figure 13.2 provides an overview of gene expression 13-16

Structure and Function of tRNA In the 1950s, Francis Crick & Mahon Hoagland proposed the adaptor hypothesis tRNAs play a direct role in the recognition of codons in the mRNA Proline anticodon

tRNA 2º Structure Figure 13.10 D loop TψC loop y = pseudouridine loop Found in all tRNAs D loop TψC loop D D The modified bases are: I = inosine mI = methylinosine T = ribothymidine D= dihydrouridine m2G = dimethylguanosine y = pseudouridine loop Structure of tRNA Figure 13.10

3º Structure of tRNA

Charging of tRNAs aminoacyl-tRNA synthetases The enzymes that attach amino acids to tRNAs There are >20 types One for each amino acid Ones for isoacceptor tRNAs put same a.a. on different tRNAs Aminoacyl-tRNA synthetases catalyze a two-step reaction 1- adenylation of amino acid 2- aminoacylation of tRNA

Aminoacyl tRNA Synthetase Function Figure 13.11 The amino acid is attached to the 3’ OH by an ester bond

tRNAs and the Wobble Rule The genetic code is degenerate There are >20 but < 64 tRNAs How does the same tRNA bind to different codons? Francis Crick proposed the wobble hypothesis in 1966 to explain the pattern of degeneracy, 1st two bases of the codon-anticodon pair strictly by Watson-Crick rules The 3rd position can wobble This movement allows alternative H-bonding between bases to form non-WC base paring

Figure 13.12 Wobble position and base pairing rules tRNAs charged with the same amino acid, but that recognize multiple codons are termed isoacceptor tRNAs Wobble position and base pairing rules Figure 13.12

Wobble Base-Pairing between anticodon & codon W-C base pairing Wobble pairing Wobble pairing

Ribosome Structure and Assembly Translation occurs on the surface of a large macromolecular complex termed the ribosome Prokaryotic cells 1 type of ribosome located in the cytoplasm Eukaryotic cells 2 types of ribosomes 1 found in the cytoplasm 2nd found in organelles -Mitochondria; Chloroplasts These are like prokaryotic ribosomes

Prokaryotic Ribosomes (a) Bacterial cell Figure 13.13

Eukaryotic Ribosomes Figure 13.13

Functional Sites of Ribosomes During bacterial translation, the mRNA lies on the surface of the 30S subunit As a polypeptide is being synthesized, it exits through a hole within the 50S subunit Ribosomes contain three discrete sites Peptidyl site (P site) Aminoacyl site (A site) Exit site (E site) Ribosomal structure is shown in Figure 13.14 13-57 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Figure 13.14

Stages of Translation Initiation Elongation Termination

Stages of Translation Initiator tRNA Release factors Figure 13.15

Translation Initiation Components mRNA, initiator tRNA, Initiation factors ribosomal subunits The initiator tRNA In prokaryotes, this tRNA is designated tRNAifmet It carries a methionine modified to N-formylmethionine In eukaryotes, this tRNA is designated tRNAimet It carries an unmodified methionine In both cases the initiator tRNA is different from a tRNAmet that reads an internal AUG codon

Prokaryotic Ribosome-mRNA Recognition 16S rRNA binds to an mRNA at the ribosomal-binding site or Shine-Dalgarno box 7 nt 16S rRNA Figure 13.17

Prokaryotic Translation Initiation (actually 9 nucleotides long) Figure 13.16

Prokaryotic Translation Initiation The tRNAiMet is positioned in the P site All other tRNAs enter the A site Figure 13.16

Eukaryotic mRNA-Ribosoime Recognition In eukaryotes, the assembly of the initiation complex is similar to that in bacteria However, additional factors are required Note that eukaryotic Initiation Factors are denoted eIF Refer to Table 13.7 The initiator tRNA is designated tRNAmet It carries a methionine rather than a formylmethionine 13-65 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

Eukaryotic Ribosome Binding The consensus sequence for optimal start codon recognition is show here Start codon G C C (A/G) C C A U G G -6 -5 -4 -3 -2 -1 +1 +2 +3 +4 Most important positions for codon selection This sequence is called Kozak’s consensus after Marilyn Kozak who first determined it

Eukaryotic Translation Initiation Initiation factors bind to the 5’ cap in mRNA & to the pA tail These recruit the 40S subunit, tRNAimet The entire assembly scans along the mRNA until reaching a Kozak’s consensus Once right AUG found, the 60S subunit joins Translation intitiates

Translation Elongation During this stage, the amino acids are added to the polypeptide chain, one at a time The addition of each amino acid occurs via a series of steps outlined in Figure 13.18 This process, though complex, can occur at a remarkable rate In bacteria  15-18 amino acids per second In eukaryotes  6 amino acids per second

Translation Elongation – tRNA Entry A charged tRNA binds to the A site EF-1 facilitates tRNA entry The 23S rRNA (a component of the large subunit) is the actual peptidyl transferase Peptidyl transferase catalyzes peptide bond formation The polypeptide is transferred to the aminoacyl-tRNA in the A site Thus, the ribosome is a ribozyme! Figure 13.18

Translation Elongation -Translocation The ribosome translocates one codon to the right promoted by EF-G uncharged tRNA released from E site The process is repeated, again and again, until a stop codon is reached Figure 13.18

Translation Termination Occurs when a stop codon is reached in the mRNA Three stop or nonsense codons UAG UAA UGA Recognized by proteins called release factors – NOT tRNAs

Translation Termination Bacteria have three release factors RF1 - recognizes UAA and UAG RF2 - recognizes UAA and UGA RF3 - binds GTP and facilitates termination process Eukaryotes only have one release factor eRF1 - recognizes all three stop codons

Translation Termination Ribosomal subunits & mRNA dissociate Figure 13.19

Polypeptides Have Directionality Translation begins at 5’ end of mRNA 5’3’ Peptide bonds are formed directionally Peptide bond is formed between the COO- of the previous amino acid in the chain and the NH2 of the amino acid being added

Peptide Bond Formation Carboxyl group Amino group Figure 13.20

Colinearity of DNA, mRNA, & Protein Sequence N terminal C terminal Figure 13.20

The amino acid sequence of the enzyme lysozyme Within the cell, the protein will not be found in this linear state It will adapt a compact 3-D structure Indeed, this folding can begin during translation The progression from the primary to the 3-D structure is dictated by the amino acid sequence within the polypeptide The amino acid sequence of the enzyme lysozyme 129 amino acids long Figure 13.4

A protein subunit Figure 13.6

Molecular Basis of Phenotype