1 TRANSLATION

2 TRANSLATION INTRODUCTION Protein (amino acid polymer, N terminus, C terminus Ribosomes: 30 S subunit plus 50 S subunit form functional 70 S ribosome mRNA is read 5' to 3' Initiation Codons; Stop Codons Polysomes INITIATION Initiator tRNA (N - formyl methionyl tRNA) Initiation complex ELONGATION Peptidyl transferase A, P, E sites on ribosome TERMINATION Stop codons SHINE-DALGARNO

AMIDES 3 PEPTIDE BOND AMINE ACID AMIDE MONO SUBSTITUTED DISUBSTITUTED PEPTIDE-AMIDE FORMED BETWEEN TWO AMINO ACIDS: ALANYL-GLYCINE PEPTIDE BOND

[STOP CODONS = UAA/UAG/UGA] 4 PROMOTER +1 GENE 5' 3' 3' 5' TRANSCRIPTION 5' 3' UNTRANSLATED REGION TRANSLATION N C CODE NOBEL HOLLEY NIRENBERG KHORANA [STOP CODONS = UAA/UAG/UGA]

RIBOSOMES - NON-SPECIFIC 5 RIBOSOMES - NON-SPECIFIC TRANSLATE ANY MESSAGE PROKARYOTIC 70S 50S + 30S SUBUNITS COMPOSITION 50S 30S NUMBER OF DIFFERENT PROTEINS 34 21 NUMBER OF DIFFERENT rRNA MOLECULES ONE - 5S ONE - 23S ONE 16S POLYSOMES

EACH SPECIFIC FOR 1 AMINO ACID AND 1 CODON 6 TRANSFER RNA tRNA ~ 60 EACH SPECIFIC FOR 1 AMINO ACID AND 1 CODON HO 3' 5' tRNAPHE SPECIFIC FOR PHENYLALANINE 3' 5' ANTICODON 5' 3' mRNA CODON FOR PHENYLALANINE tRNA - IS AN ADAPTER - ADAPTS AMINO ACID AND CODON

AMINO ACID + ATP + tRNA AMINO ACYL-tRNA + AMP + PPi 8 OVERALL: AMINO ACID + ATP + tRNA AMINO ACYL-tRNA + AMP + PPi STEP 1: + NUCLEOTIDYL TRANSFER ANHYDRIDE PPi AMINO ACID + ATP AMINO ACYL-AMP + PPi AMINO ACID ACTIVATING ENZYME = AMINO ACYL~tRNA SYNTHETASE

STEP 2: AMINO ACYL AMP + tRNA AMINO ACYL~tRNA + AMP 9 STEP 2: AMINO ACYL AMP + tRNA AMINO ACYL~tRNA + AMP AMINO ACYL~AMP 3' END tRNA AMINO ACYL~tRNA AMP

A THREONINE tRNA WITH AN ANTI-CODON OF 7 GENETIC CODE IS DEGENERATE - DIFFERENT TRIPLETS ENCODE THE SAME AMINO ACID ACA ACG ACC ACU ALL CODE THREONINE "WOBBLE" MEANS THAT SOME SPECIFIC tRNAs CAN READ MORE THAN ONE CODON A THREONINE tRNA WITH AN ANTI-CODON OF 3' UGG 5' 5' ACC 3' 5' ACU 3' PAIRS WITH OR THREONINE "WOBBLE" MAKES IT POSSIBLE FOR AN ORGANISM TO TRANSLATE ALL 61 CODONS WITH FEWER THAN 61 tRNAs PROKARYOTES OFTEN HAVE 60 - 80 tRNAs SOME DIFFERENT tRNAs CARRY THE SAME AMINO ACIDS SOME HIGHLY SPECIALIZED PROKARYOTES HAVE ~ 40 tRNA GENES

INITIATING TRANSLATION 9 INITIATING TRANSLATION FORMYL METHIONINE~tRNA [SPECIFIC FOR STARTING TRANSLATION] METHIONINE + tRNAfMET + ATP + ACTIVATING ENZYME fMET fMET FORMYL GROUP [FOLIC ACID] N-FORMYL METHIONYL ~tRNAfMET METHIONYL tRNAfMET

INITIATION COMPLEXES 10 mRNA + 30S + F - MET~tRNAfMET + GTP + INITIATION PROTEINS F MET 30S INITIATION COMPLEX 30S SUBUNIT 5' pppG mRNA 3' F MET 50S SUBUNIT 70S RIBOSOME 3' pppG E P A EXIT ACCEPTOR PEPTIDYL

ELONGATION-POLYMERIZATION 11 ELONGATION-POLYMERIZATION F-MET SER 5' G 3' P A F-MET SER TYR 5' G 3' P A PEPTIDYL TRANSFERASE [CHLORAMPHENICOL]

12 5' 3' P A TRANSLOCATION [ERYTHROMYCIN – INHIBITS MOVEMENT F-MET HO SER TYR 5' 3' P A TRANSLOCATION [ERYTHROMYCIN – INHIBITS MOVEMENT AMINO ACYL TRNA FROM A SITE TO P SITE, BLOCKS ELONGATION; AZITHROMYCIN] F-MET HO SER TYR 5' 3' E P A

GUC 13 HO GLN F-MET SER HO TYR 5' E A P TETRACYCLINE – INHIBITS AMINO ACYL TRNA BINDING A SITE DOXYCYCLINE - NEWER STREPTOMYCIN - TRNA MISMATCHING, MIS-TRANSLATION SPECTINOMYCIN - BINDS 30S SUBUNIT; EXACT MECHANISM ? ALL INHIBIT 30S RIBOSOME FUNCTION

PEPTIDYL TRANSFERASE PEPTIDYL TRANSFERASE UN-CHARGED tRNA 14 PEPTIDYL TRANSFERASE UN-CHARGED tRNA PEPTIDYL ~ tRNA

AUG AUG UAA HOW DO RIBOSOMES KNOW WHERE TO START TRANSLATING? AUG AUG 15 HOW DO RIBOSOMES KNOW WHERE TO START TRANSLATING? A. HOW TO DISTINGUISH INITIAL AUG FROM INTERNAL AUG AUG AUG UAA 5' 3' PROTEIN CODING REGION UNTRANSLATED REGION INTERNAL AUG B. HOW TO RECOGNIZE SEVERAL INITIATING AUG's IN ONE mRNA AUG AUG AUG 5' 3' 1st CODING REGION 2nd CODING REGION 3rd CODING REGION POLY CISTRONIC mRNA RIBOSOMES TRANSLATE ALL THREE INDEPENDENTLY. HOW?

SHINE DALGARNO SEQUENCES: 16 SHINE DALGARNO SHINE DALGARNO AUG AUG 5' 3' CODING REGION CODING REGION PROTEIN CODING REGIONS ARE PRECEDED BY SHINE-DALGARNO SEQUENCES [PROKARYOTES] SHINE DALGARNO SEQUENCES: ~ 7 +1 5' SHINE DALGARNO

17 BINDING BETWEEN SHINE-DALGARNO AND rRNA (16S) POSITIONS mRNA SO THAT AUG OCCUPIES P SITE 30S SUBUNIT SITES

18 REPLICATION, TRANSCRIPTION & TRANSLATION - DYNAMIC mRNA RIBOSOMES PROTEINS THEY ALL GO ON AT THE SAME TIME !

RIBOSOMES ARE RIBOZYMES - THE ACTIVE SITE FOR CATALYZING PEPTIDE BOND FORMATION IS ENTIRELY RNA 23S rRNA IS PEPTIDYL TRANSFERASE ADENINE [#2451] PARTICIPATES IN FORMING PEPTIDE BONDS 19 CHLORAMPHENICOL BINDS A 2451 & A 2452 - INHIBITS ADENINE #2451 APPRECIATE ! H BOND ADENINE N3 TAKES PROTON NITROGEN ATTACKS

20 APPRECIATE ! tRNA OXYGEN TAKES PROTON tRNA PEPTIDE BOND BREAKS H BOND STABILIZES INTERMEDIATE tRNA OXYGEN TAKES PROTON tRNA PEPTIDE BOND BREAKS OXYANION ELECTRONS FORM C O APPRECIATE !

GROWING PEPTIDE HAS BEEN ADDED TO ONE MORE AMINO ACID 21 PEPTIDE BOND FORMS APPRECIATE ! GROWING PEPTIDE HAS BEEN ADDED TO ONE MORE AMINO ACID OR; ONE MORE AMINO ACID HAS BEEN ADDED TO GROWING PEPTIDE ADENINE #2451 IS NOW READY TO REPEAT THE PROCESS