1. Lecture 2: Bacterial Polymerization Reading assignments in Text: Lengeler et al. 1999 Text: pages 343-352 DNA replication Text: pages 362-368, 441 RNA transcription Text: pages 369-376 Translation Lecture 1 Reading assignments in Text: Lengeler et al. 1999 Text: pages 110-113 Metabolic overview Text: pages 568-569, 573-574 Pili (Fimbriae) and flagella Text: pages 825-829 Surface virulence factors

2. Recap and prospectus Assembly Fuelling Biosyn. Polymer. Lecture 1 Pili Flagella Lecture 2 Lectures 3,4 Pili = “extra-cellular microtubules” pap system illustrated: Protein chaperones (PapD) Ordered substrate production Reaction BCA Substrate Y. pestis illustrated: Flagella = + Pump Turbine+ Propeller Salmonella illustrated: Check-point control (Sigma:Anti-sigma) Metabolism = Type III secretion

3. Replication DNA, RNA, Protein polymerization Biological polymerization = Assembly (+catalysis) DNARNAProteinFunction TranscriptionTranslation ?What makes a good drug ? Antibiotic ? BacteriaPeople

4. Replicating cell DNA (chromosome) replication origin forks Precise Processive(clamped from origin to end) Factory model (proofreading, <1 error/ 4x10 bp) 6 Rep- GFP Resting cell

5. Replication forks 5’ 3’ DnaB (helicase) 5’ Leading strand DNA Pol III DnaN (clamp) RNA primer Okazaki fragments on lagging strand DNA Pol III Clamp loading complex Primase DNA Pol I DNA Gyrase

6. Back DNA Gyrase A2B2A2B2 Tyr~DNA Front Grab Cut/ hold Ligate -G-G Tension at forks ATP Only bacterial topo-isomerases A E. coli B Sub-units A Yeast Topo 2 B Nalidixic acid (Nx) Novobiocin (Nov) Nx Nov

7. Topo-isomerases, Gyrase and Topo IV or “give me a break …” DNA Type II Type I E. coli (most bacteria): Topo-isomerases:Type I TopA TopB Type II Gyrase predicted “swivel” from replication fork model major replication “swivel” chromosome partition ParC, ParD ParA, ParB Replication fork tension Chromosome partition/ separation Gyrase Topo IV Nx, Nov Biologic function ? Always essential Topo IV cis-grab trans-grab

8. Bacterial transcription RNA polymerases in the 3 Kingdoms Eubacteria Archae bacteriaEukaryotes   ’ A’ B’ A B 2x  Catalytic sub-units >10 Weak homology Strong homology “Core” “TATA box factors” + many others  Sigma factorsDNA recognition  28 flagellin genes  32 Heat shock  54 Nitrogen assimilation  S Stationary “growth phase”  70 most genes e.g. E. coli Rifampicim (Rif) blocks initiation Rif

9. Transcription cycle in Eubacteria  sigma DNA ~40 bp promoter terminator ppp ribosome ppp uuuu 3’ “hair-pin” rho -independent termination Core polymerase rho factor termination eject = RNA/DNA helicase Rifampicim (Rif) blocks initiation Rif plug in RNA cleft 5’ RNA

10. Translation (protein synthesis) ppp Proteins are made on ribosomes Programmed by mRNA tRNA’s decode the “genetic code” tRNA’s mediate between the RNA and Protein “worlds” 3’ = ~ A ~ A ~ A ATP “charged” Amino acid with high energy bond Anti-codon Aminoacyl tRNA Synthetases >20 Synthetases Proofread Amino acids 50 S 5’ mRNA 30 S ppp

11. mRNA alignment by the Shine & Dalgarno sequence 5’ mRNA 30 S ? 5’ mRNA UAUCCGAUUAAGGAACGACCAUGACGCAA... 16 S RNA 3’ 16 S RNA HO-AUUCCUCCAC... Protein start Shine & Dalgarno E. coli Start codons ~90% AUG ~9% GUG ~1% UUG Protein coding Methionine Valine Leucine Starting amino acid f Met Uniquely eubacterial f Met cutting Innate immunity receptors Phylogeny standard

12. Adenylation Antibiotics target translation Antibiotic 1 Streptomycin 2 Tetracycline 3 Chloramphenicol 4 Erythromycin Blocks Initiation Elongation Binds 30 S 50 S Prevents mRNA binding f Met~tRNA binding Peptide bond formation Ribosome translocation ? Source of antibiotics: Streptomyces sps. Resistance genes / counteractions R-plasmids Counteractions acetylation Pump Counteraction (and resistance genes target antibiotics) Adenylation

13. Initiation of translation Inactive Initiation Factors IF1, 2, 3 GTP 5’ mRNA GTP ~ M f Met~tRNA ~ M GDP + P i ~ M 70 S complex 1 Streptomycin 2 Tetracycline Strep Tet Both block assembly reactions

14. Translocation Translation elongation ~ P A ~ ~ P A ~ EF-Tu GTP EF-G GTP ~ AA~ tRNA binding Peptide bond formation -  G from Pep ~ tRNA (ATP) n n +1 N-terminus 3 Chloramphenicol 4 Erythromycin rRNA catalysis “RNA world”

15. Almost THE END, Translation termination ~ Stop codonsUAA UAG UGA Release Factors ( 3 RF proteins) GTP ATP GTP Peptide bonds (+ biosyn.) Translation / assembly reactions -  G “division of labor”

16. Polymerization without a nucleus DNA RNA protein membrane Who needs a nucleus ? Bacteria >10 x higher protein synthesis rates “prokaryotes” vs “eukaryotes” During rapid growth ~50% mass = proteins syn. system 3 IF’s vs 10 IF’s Smaller ribosome = 50:50 RNA:protein Polycistronic mRNA

17. What makes a good Antibiotic ? 1 Distinguish 2 Block 3 Cause danger Major Assemblies minor assemblies General Biosyn. specific biosyn. Unique genes (most, e.g. biosyn., flagella) Gene families (PBP’s, Topo’s) Target heirarchy