Download CHAPTER 36: RNA Synthesis and Regulation in Bacteria

CHAPTER 36: RNA Synthesis and
Regulation in Bacteria
(Problems: 1-6,10-13,15-19,25)
36.1 Cellular RNA is Synthesized by RNA Polymerases
RNA synthesis involves:
• A template (DNA)
• Activated precursors (ribo-NTPs)
• A divalent metal ion (Mg2+)
Mg2+
Types of RNA
(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
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”
• Strong
promoters
match
consensus
sequence
closely (operons
transcribed
efficiently)
• Weak
promoters
match
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
Transcription bubble
• Error rate is 10-4 – 10-5 (vs 10-7
for DNA pol.)
 Not transmitted to progeny
 Many RNA transcripts
Termination:
formation of phosphodiester bonds stop,
RNA-DNA hybrid dissociates, the melted region of DNA
reanneals, and RNA polymerase is released.
Protein independent
termination
Protein dependent
termination
• 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.
Processing or RNA Transcripts
RNase P
RNase 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 galactosidase
which serves to detect lactose when
present
• When lactose is detected,
allolactose is produced resulting in
the removal of the Lac repressor
Operon
Regulator gene
Operator site
Structural gene
Repressor
Inducer
Polycystronic
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 50fold, (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 activated
and 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