Copyright The Mc GrawHill Companies Inc Permission required

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

n Special codons: n AUG (which specifies methionine) = start codon n UAA, UAG and UGA = termination, or stop, codons The code is degenerate n More than one codon can specify the same amino acid n n For example: GGU, GGC, GGA and GGG all code for lysine In most instances, the third base is the degenerate base n n AUG specifies additional methionines within the coding sequence It is sometime referred to as the wobble base The code is nearly universal n Only a few rare exceptions have been noted n Refer to Table 13. 3 Copyright ©The Mc. Graw-Hill Companies, Inc. Permission required for reproduction or display 13 -14

n Figure 13. 2 provides an overview of gene expression Figure 13. 2 13 -16

Structure and Function of t. RNA n n In the 1950 s, Francis Crick & Mahon Hoagland proposed the adaptor hypothesis t. RNAs play a direct role in the recognition of codons in the m. RNA Proline anticodon

t. RNA 2º Structure Found in all t. RNAs D loop TψC loop D D n n n n Figure 13. 10 Structure of t. RNA loop The modified bases are: I = inosine m. I = methylinosine T = ribothymidine D= dihydrouridine m 2 G = dimethylguanosine y = pseudouridine

3º Structure of t. RNA

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

Aminoacyl t. RNA Synthetase Function Figure 13. 11 The amino acid is attached to the 3’ OH by an ester bond

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

t. RNAs charged with the same amino acid, but that recognize multiple codons are termed isoacceptor t. RNAs Figure 13. 12 Wobble position and base pairing rules

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

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

Prokaryotic Ribosomes (a) Bacterial cell Figure 13. 13

Eukaryotic Ribosomes Figure 13. 13

Functional Sites of Ribosomes n During bacterial translation, the m. RNA lies on the surface of the 30 S subunit n n Ribosomes contain three discrete sites n n As a polypeptide is being synthesized, it exits through a hole within the 50 S subunit Peptidyl site (P site) Aminoacyl site (A site) Exit site (E site) Ribosomal structure is shown in Figure 13. 14 Copyright ©The Mc. Graw-Hill Companies, Inc. Permission required for reproduction or display 13 -57

Figure 13. 14

Stages of Translation n Initiation Elongation Termination

Stages of Translation Initiator t. RNA Release factors Figure 13. 15

Translation Initiation n Components n n n m. RNA, initiator t. RNA, Initiation factors ribosomal subunits The initiator t. RNA n In prokaryotes, this t. RNA is designated t. RNAifmet n n In eukaryotes, this t. RNA is designated t. RNAimet n n It carries a methionine modified to N-formylmethionine It carries an unmodified methionine In both cases the initiator t. RNA is different from a t. RNAmet that reads an internal AUG codon

Prokaryotic Ribosome-m. RNA Recognition n 16 S r. RNA binds to an m. RNA at the ribosomal-binding site or Shine-Dalgarno box 7 nt Figure 13. 17 16 S r. RNA

Prokaryotic Translation Initiation (actually 9 nucleotides long) Figure 13. 16

Prokaryotic Translation Initiation The t. RNAi. Met is positioned in the P site All other t. RNAs enter the A site Figure 13. 16

Eukaryotic m. RNA-Ribosoime Recognition n In eukaryotes, the assembly of the initiation complex is similar to that in bacteria n However, additional factors are required n n n Note that eukaryotic Initiation Factors are denoted e. IF Refer to Table 13. 7 The initiator t. RNA is designated t. RNAmet n It carries a methionine rather than a formylmethionine Copyright ©The Mc. Graw-Hill Companies, Inc. Permission required for reproduction or display 13 -65

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

Eukaryotic Translation Initiation n n Initiation factors bind to the 5’ cap in m. RNA & to the p. A tail These recruit the 40 S subunit, t. RNAimet The entire assembly scans along the m. RNA until reaching a Kozak’s consensus Once right AUG found, the 60 S subunit joins Translation intitiates

Translation Elongation n 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 n n In bacteria 15 -18 amino acids per second In eukaryotes 6 amino acids per second

Translation Elongation – t. RNA Entry n n A charged t. RNA binds to the A site EF-1 facilitates t. RNA entry The 23 S r. RNA (a component of the large subunit) is the actual peptidyl transferase Thus, the ribosome is a ribozyme! Figure 13. 18 n n Peptidyl transferase catalyzes peptide bond formation The polypeptide is transferred to the aminoacyl-t. RNA in the A site

Translation Elongation Translocation n n The ribosome translocates one codon to the right promoted by EF-G n n Figure 13. 18 uncharged t. RNA released from E site The process is repeated, again and again, until a stop codon is reached

Translation Termination n n Occurs when a stop codon is reached in the m. RNA Three stop or nonsense codons n n UAG UAA UGA Recognized by proteins called release factors – NOT t. RNAs

Translation Termination n Bacteria have three release factors n n RF 1 - recognizes UAA and UAG RF 2 - recognizes UAA and UGA RF 3 - binds GTP and facilitates termination process Eukaryotes only have one release factor n e. RF 1 - recognizes all three stop codons

Translation Termination Ribosomal subunits & m. RNA dissociate Figure 13. 19

Polypeptides Have Directionality n Translation begins at 5’ end of m. RNA n n n 5’ 3’ Peptide bonds are formed directionally Peptide bond is formed between the COO- of the previous amino acid in the chain and the NH 2 of the amino acid being added

Peptide Bond Formation Carboxyl group Figure 13. 20 Amino group

Colinearity of DNA, m. RNA, & Protein Sequence N terminal Figure 13. 20 C terminal

n The amino acid sequence of the enzyme lysozyme Within the cell, the protein will not be found in this linear state n It will adapt a compact 3 -D structure n 129 amino acids long Figure 13. 4 n 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

A protein subunit Figure 13. 6

Molecular Basis of Phenotype