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

Central Dogma DNA -> RNA -> Protein

STRUCTURE OF DNA Fig. 10.1

Bases and Sugars of DNA and RNA

Base-pairing

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.

Supercoiling

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

Topoisomerases

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’

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

Semi-conservative Replication Fig. 10.3

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

Fig. 10.9

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.  -clamp loading: a ring-shaped homodimer encircles DNA strands to aid binding of DNA polymerase III.

Activities at the Fork 5’ 3’ Fig

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

Action of DNA ligase

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

Termination of Replication

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

Bacterial RNA polymerase Responsible for synthesis of all 3 types of RNA species Huge enzyme (400 kD) made of five subunits: 2  subunits 1  subunit 1  ’ subunit 1  factor core enzyme holoenzyme

Promoter structure

Transcription Initiation

Fig

Elongation (polymerization)

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

Transcription termination Fig

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

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

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

Fig