CHAPTER 36: RNA Synthesis andRegulation in Bacteria(Problems: 1-6,10-13,15-19,25)

RNA synthesis involves:• A template (DNA)• Activated precursors (ribo-NTPs)• A divalent metal ion (Mg2+)

36.1 Cellular RNA is Synthesized by RNA Polymerases

Mg2+

(1) Transfer RNA (tRNA)• Carries amino acids to translation machinery• Very stable molecules

(2) Ribosomal RNA (rRNA) • Makes up much of the ribosome• Very stable, majority of cellular RNA

(3) Messenger RNA (mRNA) • Encodes message from DNA to ribosomes• Rapidly degraded by nucleases

Types of RNA

RNA Synthesis

• Growing RNA chain is base-paired to DNA template strand• Catalyzes polymerization in 5’3’ direction• Is highly processive, and thermodynamically assisted by PPi hydrolysis• Incoming RTPs: UTP, GTP, ATP, CTP• Growing single-stranded RNA released • Adds 30-85 nucleotides/sec (~ 1/10th rate of DNA replication)

36.2 RNA Synthesis Comprises Three Stages

Initiation Elongation Termination

Initiation: How does RNA polymerase determine where to begin?

Promoters

Pribnow box“TATA”

• Strongpromotersmatch consensus sequence closely (operons transcribed efficiently)

• Weakpromotersmatch consensus sequences poorly (operons transcribed less frequently)

• protein and binding of RNA polymerase to DNA.

• protein and assembly of RNA polymerase at the “UP” site.

Elongation

• Error rate is 10-4 – 10-5 (vs 10-7

for DNA pol.)

Not transmitted to progeny Many RNA transcripts

Transcription bubble

Termination: formation of phosphodiester bonds stop, RNA-DNA hybrid dissociates, the melted region of DNA reanneals, and RNA polymerase is released.

• A palindromic GC-rich region + T’s

5’-AGCCGCCAGTTCGGCTGGCGGCATTTT-3’3’-TCGGCGGTCAAGCCGACCGCCGTAAAA-5’5’-AGCCGCCAGUUCGGCUGGCGGCAUUUU-3’

• Formation of an RNA hair-pin followed by U’s.

• Termination via the rho-protein• Rho is hexameric, binds a 72

stretch of nucleotides on RNA that are C-rich and G-poor.

• Rho moves upward on RNA driven by energy from ATP (ATPase) and pulls RNA away from DNA.

Protein independent termination

Protein dependent termination

Processing or RNA Transcripts

RNase PRNase III

+ + +

+ + +

36.3 The lac operon and Control of Bacterial Gene Expression

(1) Lactose permease (lacY gene product) - transporter for uptake of -galactosides.(2) -Galactosidase (lacZ gene product) - hydrolyzes -galactosides to hexoses.(3) Thiogalactoside transacetylase (lacA gene product) – acetylates nonmetabolizable -galactosides prior to elimination from the cell.

When glucose is present these proteins are synthesized in limited amounts

When glucose levels are low these proteins are synthesized in larger amounts

Binding of lac repressor to the lac operon• Tetrameric lac repressor (lacI

product) interacts simultaneously with two sites near the lac promoter

• DNA loop forms

• RNA pol can still bind to the promoter but cannot pass the loop

• In the presence of lactose, an inducer, allolactose, is produced by -galactosidase

• Only 10 Lac repressor per cell resulting in “escape synthesis”

• “escape synthesis” allows small level of permease and galactosidasewhich serves to detect lactose when present

• When lactose is detected, allolactose is produced resulting in the removal of the Lac repressor

OperonRegulator geneOperator siteStructural geneRepressorInducerPolycystronic

cAMP Regulatory Protein Activates Transcription

• The lac operon is transcribed maximally when -galactosides are the only carbon source

• When glucose is present transcription is reduced 50-fold, (catabolite repression)

• A weak promoter in these operons is promoted to a stronger one by an activator (in the absence of glucose)

• cAMP response (or receptor) protein (CRP) is the activator

• CRP is also known as catabolite activator protein (CAP)

• In the presence of cAMP, CRP binds near the promoters of more than 30 genes

• RNA polymerase is activatedand the rate of transcription activation is increased

• Activation of transcription initiation at the lac promoter by CRP-cAMP

• In the absence of glucose, and the presence of galactosides enzyme III (EIII) transfers a phosphate group to adenylate cyclase leading to CRP-cAMP increases

• CRP-cAMP activates transcription of other genes