1 TRANSLATION 2 TRANSLATION INTRODUCTION Protein amino acid

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 m. RNA is read 5' to 3' Initiation Codons; Stop Codons Polysomes INITIATION Initiator t. RNA (N - formyl methionyl t. RNA) Initiation complex ELONGATION Peptidyl transferase A, P, E sites on ribosome TERMINATION Stop codons SHINE-DALGARNO

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

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

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

TRANSFER RNA t. RNA ~ 60 EACH SPECIFIC FOR 1 AMINO ACID AND 1 CODON HO 3' 5' t. RNAPHE SPECIFIC FOR PHENYLALANINE 3' 5' 5' ANTICODON 3' m. RNA CODON FOR PHENYLALANINE t. RNA - IS AN ADAPTER - ADAPTS AMINO ACID AND CODON 6

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

STEP 2: AMINO ACYL AMP + t. RNA AMINO ACYL~t. RNA + AMP AMINO ACYL~AMP 3' END t. RNA AMINO ACYL~t. RNA AMP 9

7 GENETIC CODE IS DEGENERATE DIFFERENT TRIPLETS ENCODE THE SAME AMINO ACID ACA ACG ACC ACU ALL CODE THREONINE "WOBBLE" MEANS THAT SOME SPECIFIC t. RNAs CAN READ MORE THAN ONE CODON A THREONINE t. RNA WITH AN ANTI-CODON OF 3' UGG 5' PAIRS WITH OR 5' 5' ACC 3' ACU 3' THREONINE "WOBBLE" MAKES IT POSSIBLE FOR AN ORGANISM TO TRANSLATE ALL 61 CODONS WITH FEWER THAN 61 t. RNAs PROKARYOTES OFTEN HAVE 60 - 80 t. RNAs SOME DIFFERENT t. RNAs CARRY THE SAME AMINO ACIDS SOME HIGHLY SPECIALIZED PROKARYOTES HAVE ~ 40 t. RNA GENES

9 INITIATING TRANSLATION FORMYL METHIONINE~t. RNA [SPECIFIC FOR STARTING TRANSLATION] METHIONINE + t. RNAf. MET + ATP + ACTIVATING ENZYME f. MET FORMYL GROUP [FOLIC ACID] METHIONYL t. RNAf. MET N-FORMYL METHIONYL ~t. RNAf. MET

10 INITIATION COMPLEXES F MET 30 S INITIATION COMPLEX 5' ppp m. RNA + 30 S + F - MET~t. RNAf. MET + GTP + INITIATION PROTEINS 30 S SUBUNIT 3' m. RNA G F MET 50 S SUBUNIT 70 S RIBOSOME ppp 3' G EXIT E A P ACCEPTOR PEPTIDYL

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

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

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

PEPTIDYL TRANSFERASE UN-CHARGED t. RNA PEPTIDYL ~ t. RNA 14

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

16 E O IN RN SH GA L A D AUG 5' AUG CODING REGION PROTEIN CODING REGIONS ARE PRECEDED BY SHINE-DALGARNO SEQUENCES [PROKARYOTES] SHINE DALGARNO SEQUENCES: ~7 +1 5' SHINE DALGARNO 3'

BINDING BETWEEN SHINE-DALGARNO AND r. RNA (16 S) POSITIONS m. RNA SO THAT AUG OCCUPIES P SITE 30 S SUBUNIT SITES 17

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

19 RIBOSOMES ARE RIBOZYMES THE ACTIVE SITE FOR CATALYZING PEPTIDE BOND FORMATION IS ENTIRELY RNA 23 S r. RNA IS PEPTIDYL TRANSFERASE ADENINE [#2451] PARTICIPATES IN FORMING PEPTIDE BONDS CHLORAMPHENICOL BINDS A 2451 & A 2452 - INHIBITS ADENINE #2451 APPRECIATE ! H BOND ADENINE N 3 TAKES PROTON NITROGEN ATTACKS

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

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