Bacterial Physiology (Micr430)

1. Bacterial Physiology (Micr430) Lecture 8 Macromolecular Synthesis and Processing: DNA and RNA (Text Chapter: 10)

2. Central Dogma DNA -> RNA -> Protein

3. STRUCTURE OF DNA Fig. 10.1

4. Bases and Sugars of DNA and RNA

5. Base-pairing

6. Supercoiled DNA • In cells, DNA is highly compacted into tertiary structure. • Bacterial chromosome is a covalently closed, circular, double-stranded DNA molecular. • To be maximally compacted, DNA needs to be in a negatively supercoiled structure.

7. Supercoiling

8. Topoisomerases • Topoisomerases are enzymes that alter the topological form (supercoiling) of a circular DNA molecule. • Type I topoisomerases can cleave one strands of DNA; requires no ATP • Type II topoisomerases can cleave both strands of DNA; requires ATP

9. Topoisomerases

10. DNA Replication • Semiconservative replication • Bidirectional • DNA polymerase functions as a dimer • Replication non-continuous (Okazaki fragments) • Orientation of new strand synthesis is 5’ to 3’

11. Semi-conservative Replication • DNA replication proceeds in a semi-conservative manner. • This was hypothesized by Watson and Crick and experimentally confirmed by Messelson and Stahl

12. Semi-conservative Replication Fig. 10.3

13. Replication Initiation • Replication initiates at oriC locus • oriC contains several 13-mer AT-rich sequences • DnaA serves as positive regulator of initiation; it binds to five 9-mer sequences within oriC • DnaA binding to oriC promotes strand opening of the AT-rich 13-mers, facilitating the loading of DnaB helicase

14. Fig. 10.9

15. Model of DNA replication • 1. Prepriming (Primosome): DnaB, DnaC and DnaG (primase) involved • 2. Unwinding: DNA gyrase • 3. Priming: primase (DnaG) synthesizes RNA primer • 4. b-clamp loading: a ring-shaped homodimer encircles DNA strands to aid binding of DNA polymerase III.

16. Activities at the Fork 3’ 5’ 3’ 5’ Fig. 10.11

17. Model of DNA replication • 5. Completion of lagging strand: DNA pol III stops when it encounters the 5’ terminus of the previous Okazaki. • 6. Proofreading: by 3’ to 5’ exonuclease proofreading activity of DNA pol III • 7. Replacing the primer: RNAse H cleaves RNA primer and DNA Pol I fills the gap with DNA • 8. Repairing single-stranded nicks

19. Action of DNA ligase

20. Termination of Replication • Termination occurs in a region called ter • ter consists of clusters of sites called ter sequences of 22 bp long • These sites serve as one-way gates allowing replication forks to pass through in one direction but not in the other

21. Termination of Replication

22. RNA SYNTHESIS • Process is the same for synthesis of all three types of RNA • Catalyzed by RNA polymerase • Transcription consists of three main steps: • initiation • elongation • termination

23. Bacterial RNA polymerase • Responsible for synthesis of all 3 types of RNA species • Huge enzyme (400 kD) made of five subunits: • 2 a subunits • 1 b subunit • 1 b’ subunit • 1  factor holoenzyme core enzyme

24. Promoter structure

25. Transcription Initiation

26. Fig. 10.24

27. Fig. 10.24

28. Elongation (polymerization)

29. Transcription termination • Factor-independent termination • inverted repeats, forming hair-pin • short string of A’s

30. Transcription termination Fig. 10.25

31. Transcription termination • Factor-dependent termination • 3 factors • Rho (), Tau () and NusA • Rho best studied • Rho is an RNA-dependent ATPase • Also an RNA-DNA helicase • Transcription and translation is coupled in bacteria

33. RNA Turnover • Cellular RNA can be classed into 2 groups • Stable RNA: rRNA and tRNA • Unstable RNA: mRNA • Stability factors: • Ribonucleoprotein complex protects RNA • Secondary structure of RNA • Average mRNA half-life: 40 sec at 37 °C

34. Enzymes Involved • RNase P: It contains both protein and RNA components - ribozyme. Required for the maturation of tRNA. • RNase II, one of the major 3’ -> 5’ exonucleases in E. coli • RNase III, cuts dsRNA • RNase D; RNase E; RNase H; RNase R

35. Fig. 10.29