CHAPTER 40The Mechanism of Protein Synthesis

Problems: 2,3,6,7,9,13,14,15,18,19,20

• Initiation: Locating the start codon.

• Elongation: Reading the codons (5’→3’) and synthesizing protein amino→carboxyl.

• Termination: Recognizing terminal codon and releasing protein.

Prokaryotes and Eukaryotes

40:1 Decoding the Information in mRNA

Exit Peptidyl Aminoacyl

Initiation

Start codon (also GUG, Val)Shine-Delgarno Sequence

Start: 1. Pairing of 16S

rRNA with S-D of mRNA (a UTR).

2. Pairing of initiator tRNAwith start codon.

Initiator tRNAfTwo different tRNA’s that carry Met; tRNAf and tRNAm.

One Synthetase adds Met to both tRNAf and tRNAm.

A specific transformylase only formylates Met-tRNAf.

Initiation

Initiation factors, IF1, IF2 and IF3.

IF1 and IF3 prevent premature assembly of the 70S ribosome.

IF2 binds to fMet-tRNAfand mRNA at AUG codon.

The fMet-tRNAf in the fully assembled ribosome is in the P-site.

Initiation

Elongation Factor, EF-Tubrings appropriate aa-tRNAto the A-site.

fMet

AUG

Requires GTP: EF-Tu-GTPProtects aa-tRNA ester bond.

GTP→GDP only when proper codon is present and then EF-Tu is released.

40:2 Peptidyl Tranferase and Peptide Bond Formation---Elongation

Peptidyl tranferasecenter on 23S rRNAof 50S subunit.

Catalysis occurs via mechanism of proximity and orientation.

-EF-Tu-GTP-EF-Tu-GDP

EF-Tu-GDP

EF-Tu-GTP

GTP

GDPEF-Ts

Peptide grows from the H3N+-end to –COOH-end

Termination

Release Factors (RF) recognize the stop codons: RF1-UAA or UAG and RF2-UAA or UGA.

RF recognizes stop codon and interacts with peptidyltransferase promoting addition of H2O, rather than aminoacyl-tRNA, to the growing peptide chain.

GTP hydrolysis after binding EF-G and binding of ribosome release factor (RRF) cause release of ribosome.

40:3 Bacteria and Eukaryotes Differ in Initiation of Protein Synthesis

Larger ribosome: 80S versus 70S.

Initiator tRNAi and Met-tRNAi is not formylated; differs from Met-tRNAm.

Initiation involves eIF’s, CAP recognition (eIF-4E), and movement to AUG start codon (eIF-2).

Circular structure of eukaryotic mRNA thought to facilitate rebinding of ribosomes

Important control point. Mutation leads to serious disease.

Elongation: EF1 and EF1 are counterparts of EF-Tu and EF-Ts, and EF2 is equivalent of bacterial EF-G translocase.

Termination: eRF1 is the release factor and is the only release factor in eukaryotes. eIF-3 prevents premature ribosome reassembly in absence of initiation factors.

Organization: In eukaryotes the translation machinery is organized as a large complex with the cytoskeleton.

40.4: Antibiotic Protein Synthesis inhibitors.

Diphtheria toxin inhibits EF2 (the eukaryotic translocase).

40:5 Secretory and Membrane Proteins

Protein targeting or sorting: 1. Posttranslational delivery to nucleus, chloroplast, mitochondria, and peroxisomes. 2. Secretory pathway to ER, and then the Golgi, lysosomes, integral membrane proteins, etc.

Signal recognition particle

Packaged in transport vesicles and transported to location via exo- and endocytosis

40:6 Protein Synthesis RegulationRegulation by use of mRNA: Iron Response Elements (IRE)

Ferritin mRNA contains an IRE to which an IRE binding protein (IRE-BP) binds and blocks translation of the mRNA.

High iron: IRE-BP binds iron and can not bind ferritin mRNA. Ferritin is produced to store iron.

Low iron: IRE-BP binds ferritin mRNA and blocks ferritin production

Transferrin-receptor mRNA contains an IRE to which an IRE binding protein (IRE-BP) binds under low iron that stabilizes the mRNA, permitting translation and production of the receptor.

Regulation Through Small RNAsRNA interference, RNAi: External source of dsRNA is cleaved into 21-nucleotide fragments by an RNase → ss siRNA → RISC which locats siRNA onto complementary mRNA and promotes their degradation.

MicroRNAs, miRNA: miRNA produced from endogenous encoded RNA precursor this binds to complementary region of mRNA forming a 21-nucleotide ds RNA region which promotes degradation of that mRNA. Human genome has more that 700 miRNAs, 60% of genes regulated by one or more miRNAs: cell differentiation, development, disease such as cancer, etc.