Bacterial Physiology (Micr430)

Lecture 8Macromolecular Synthesis and

Processing: DNA and RNA

(Text Chapter: 10)

Central Dogma

DNA -> RNA -> Protein

STRUCTURE OF DNA

Fig. 10.1

Bases and Sugarsof 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’

5’

3’

3’

Fig. 10.11

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

coreenzyme holoenzym

e

Promoter structure

Transcription Initiation

Fig. 10.24

Fig. 10.24

Elongation (polymerization)

Transcription termination

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

Transcription termination

Fig. 10.25

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. 10.29