1 An overview of protein synthesis via transcription and translation References: 1.Genes VIII, by Lewin, 2004, Oxford. 2.Molecular Biology, by Weaver, 3rd ed.,2004, McGraw-Hill.

2 Prokaryotic Gene Expression Transcription (RNA polymerase) Translation (Ribosome) P romoter Terminator Ribosome- binding site Start codonStop codon ORF mRNA Protein

3 Transcription in Prokaryotic Cells RNA polymerase DNA template Coding strand Template strand promoter; terminator RNA pol

4 Template recognition – Initiation – Elongation - Termination 5’ 3’ Stages of transcription

5 RNA pol in eubacteria core:  2  ’ holoenzyme: core +  factor  factor is separated from the core when holoenzyme is subjected to an anion exchange (e.g. phosphocellulose) column

6 Yeast RNA polymerase RNA DNA

7 Promoter recognition. Promote tight binding of holoenzyme to the promoter. Loosening non- specific interaction between RNA pol and template. Stimulates transcription initiation. Effect of  factor Functions of  factor

8  converts a loosely bound RNA pol in a closed complex to the tightly bound pol in the open promoter complexes. Supercoiled DNA is a better template for transcription, because it requires less free energy for the initial melting of DNA. RNA pol-promoter binding

9

10 Initiation 1. Forming the closed promoter complex 2. Forming the open promoter complex 3. Abortive initiation 4. Promoter clearance Template recognition

11 Sliding along DNA does not occur How RNA polymerase gets to the promoter?

12 +Rifampicin -Rifampicin Rif R Rif S Sigma cycle  factor can be reused

13 DNA region covered by holoenzyme is from -55 to +20; that covered by core enzyme after loss of  is from -35 to +20. RNA Pol-Promoter Interaction

14 DNA footprinting

15

16 RNA Pol-Promoter Interaction Methylation Interference Assay Bases on either the template or the non- template strand that are more methylated in the filtrate than in the filter- bound DNA are presumably important in polymerase binding to the promoter.

17 RNA pol-promoter contact -9 to +3

18 Features of bacterial promoters Consensus TTTACA TTGACA TTGATA TTGACA TATGTT TTAACT GATACT TATAAT TATGTT TTGACA TATAAT 18 bp 17 bp 9 bp 18 bp 7 bp lactrp lac trp lP L recA tacI -35 >90% of transcription start point is a purine (16-19 bp) (5-9 bp)

19 How many kinds of  factors are there in a bacterial cell? What is the structure of  factor?

20

21 Primary  factors (e.g.  70 of E. coli;  43 of B. subtilis ) Alternative  factors Transcription of specialized genes (e.g.  54 of E. coli) Structure of  factors Free  cannot bind to the promoter (The N-terminal region suppresses the DNA-binding region). Only when it is bound with the core, upon which its conformation changes, can  binds the promoter.

22 Region 1. Present only in primary . The 245 aa existing in  70, but not in  43, may be involved in loosening binding between RNA pol and non-promoter regions. Region 2. Most highly conserved. 2.1 and 2.2: hydrophobic; binding to pol core. 2.3: involved in DNA melting. 2.4:  -helix; recognition of -10 box. Region 3. Helix-turn helix DNA-binding domain. Region : helix-turn-helix loop; binding to -35 box.  aa deletion

23  54 is different from other  factors in: 1.The “-35 box” is located 6 bp upstream of the “-10 box”; 2.Sites that are rather distant from the promoter influence its activity (recognized by an enhancer-binding protein); 3.The free form can bind to DNA. Different  factors recognize promoters with different consensus sequences Primary vs. alternative  factors in E. coli

24 Sigma-switching model Temporal control of transcription of B. subtilis phage SPO1

25 Other examples : Control of transcription during sporulation in B. subtilis Regulation of glutamine synthetase gene (  54 ) Regulation of the E. coli heat shock genes (  32 ) Stress-resistance genes turned on in the stationary phase (  s ) Genetic evidence Isolation of mutants that are unable to do transcription switch. Biochemical evidence Composition analysis of the RNA pol isolated from different stages.

26 Functions of RNA pol core 1.To unwind and rewind DNA 2.To hold the separated strand of DNA and RNA 3.To catalyze the addition of ribonucleotides to the growing RNA chain 4.To adjust the difficulties in processing by cleaving the RNA product and restarting RNA synthesis (with the assistance of some accessory factors, e.g., GreA and GreB in E. coli) Elongation

27 Recover of RNA polymerase from pausing

28 Role of  subunit in UP element recognition: 1. Addition of UP to the core promoter increases in vitro transcription by RNA pol alone. 2. The 94 C-terminal aa are required for UP recognition. Function of  -subunit Core enzyme assembly; Promoter recognition; Interaction with some regulators.

29 UP element: an AT-rich sequence which stimulates transcription of the rrnB gene by a factor of 30. Fis sites: binding sites for Fis, a transcriptional activator.

30  -subunit Phosphodiester bond formation. (Confers both the rifampicin- and streptolydigin-resistance) Stabilizing RNA pol-DNA complex during elongation. Forms both the salt-sensitive and salt-resistant contact with the DNA template.  ’-subunit Most basic subunit. Strongest DNA- binding activity. Forms salt-resistant contact with the DNA template. Function of  and  ’-subunit

31 The strain of unwinding is relaxed by the topoisomerases. Topology of elongation

32 Termination Mechanism  -independent termination (intrinsic terminators) Requires: a hairpin loop a string of Ts following the hairpin.

33 Intrinsic terminators Stem of hairpin: G-C-rich; 7-20 bp Loop: 5 bp or up Distance between hairpin and U-run: 7-9 bp

34 Requires a hairpin loop and  factor acts as a homohexamer each subunit contains an RNA-binding domain an ATPase domain. an RNA helicase (separates RNA-DNA hybrid)  -dependent termination Half of E. coli terminators; most are found in phage genomes

35  -dependent terminator bases long C-rich/G poor

36 Polar effect on transcription of the downstream genes caused by a nonsense mutation

37 Negative control: Positive control: Control of prokaryotic transcription Inducible genes v.s. Constitutive genes Repressor Activator

38 Cofactors of the regulators: Repressor Inducer Corepressor Activator Inducer Common features of the cofactors: Highly specific Not necessarily interact with the target enzyme Gratuitous inducers (e.g., IPTG) Allosteric control of the regulator Other positive control mechanisms: Substitution of  factors Antitermination Other means of activation of regulators: Phosphorylation Oxidation

39 4 Operon A group of contiguous, coordinately controlled genes Polycistronic mRNA The first operon discovered (Jacob and Monod, 1961) The lac operon