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

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

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

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

13-16Figure 13.2

Figure 13.2 provides an overview of gene expression

In the 1950s, Francis Crick & Mahon Hoagland proposed the adaptor hypothesis

tRNAs play a direct role in the recognition of codons in the mRNA

Structure and Function of tRNA

Proline anticodon

Structure of tRNAFigure 13.10

Found in all tRNAstRNA 2º Structure

The modified bases are: I = inosine mI = methylinosine T = ribothymidine D= dihydrouridine m2G = dimethylguanosine = pseudouridine

D loopD

D

TψC loop

loop

3º Structure of tRNA

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

Charging of tRNAs

Figure 13.11

Aminoacyl tRNA Synthetase

Function

The amino acid is attached to the 3’ OH

by an ester bond

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

tRNAs and the Wobble Rule

Wobble position and base pairing rulesFigure 13.12

tRNAs charged with the same amino acid, but that recognize multiple codons are

termed isoacceptor tRNAs

Wobble Base-Pairing between anticodon & codon

W-C base pairing

Wobble pairing

Wobble pairing

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

Ribosome Structure and Assembly

Figure 13.13

(a) Bacterial cell

Prokaryotic Ribosomes

Figure 13.13

Eukaryotic 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

Functional Sites of Ribosomes

13-57

Figure 13.14

Stages of Translation

Initiation Elongation Termination

Release factors

Initiator tRNA

Stages of Translation

Figure 13.15

Components mRNA, initiator tRNA, Initiation factors ribosomal subunits

The initiator tRNA In prokaryotes, this tRNA is designated tRNAi

fmet 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

Translation Initiation

16S rRNA binds to an mRNA at the ribosomal-binding site or Shine-Dalgarno box

16S rRNAFigure 13.17

Prokaryotic Ribosome-mRNA Recognition

7 nt

(actually 9 nucleotides long)

Figure 13.16

Prokaryotic Translation Initiation

Figure 13.16

The tRNAiMet is

positioned in the P site

All other tRNAs enter the A site

Prokaryotic Translation Initiation


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

Eukaryotic mRNA-Ribosoime Recognition

13-65

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 Ribosome Binding

Eukaryotic Translation Initiation Initiation factors bind to the 5’ cap in mRNA &

to the pA tail These recruit the 40S subunit, tRNAi

met

The entire assembly scans along the mRNA until reaching a Kozak’s consensus

Once right AUG found, the 60S subunit joins Translation intitiates

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

Figure 13.18

Translation Elongation – tRNA Entry

A charged tRNA binds to the A site

EF-1 facilitates tRNA entry

Peptidyl transferase catalyzes peptide bond formation

The polypeptide is transferred to the aminoacyl-tRNA in the A site

The 23S rRNA (a component of the large subunit) is the actual

peptidyl transferase

Thus, the ribosome is a ribozyme!

Figure 13.18

The ribosome translocates one codon to the right

promoted by EF-G

Translation Elongation -

Translocation

uncharged tRNA released from E site

The process is repeated, again and again, until a stop codon is reached

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


Ribosomal subunits & mRNA dissociate

Figure 13.19


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

Polypeptides Have Directionality

Carboxyl group Amino group

Peptide Bond Formation

Figure 13.20

Figure 13.20

N terminal C terminal

Colinearity of DNA, mRNA, & Protein Sequence

Figure 13.4

The amino acid sequence of the

enzyme lysozyme

129 amino acids long

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

Figure 13.6

A protein subunit

Molecular Basis of Phenotype

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