Chapter 17 - Prokaryotic Transcription Flashcards
Transcription proceeds in a … direction down a template strand that is oriented …
5’ to 3’
3’ to 5’
RNA polymerase
An enzyme that synthesizes a complementary RNA using a DNA template
Transcription unit
Stretch of DNA that codes for an RNA molecule and any sequences needed for transcription
RNA polymerase separates the two strands of DNA in a
transient transcription bubble
Bacterial RNA polymerase transcribes how many nucleotides per second
40-50
a single type of RNA polymerase produces what in bacteria
all RNA
holoenzyme
The form of RNA polymerase that is used to initiate transcription
holoenzyme contents
five subunits of the core polymerase and the sigma factor
RNA polymerase catalysis is derived from
the beta and beta prime subunits
the c-terminal domain in RNA polymerase
has the alpha subunits
is involved in stimulating transcription in prokaryotes
RNA polymerase has a general affinity for DNA because of
electrostatic interactions
The core polymerase can synthesize RNA, but cannot recognize
promotors
The sigma factor is used to initiate transcription at
specific sites
promotor strength
Efficiency of individual promoters in initiating transcription
Frequency of initiation varies from 1/sec to 1/30 minutes
first way RNAP finds promotors
Random diffusion and nonspecific binding to short sequences
Rapid dissociation of enzyme and repositioning
Very, very inefficient mechanism
second way RNAP finds promotors
Nonspecific binding to genome and then movement along genome to specific promoter(s)
Sliding
Intersegment transfer
Intradomain association and dissociation (hopping)
holoenzyme structure - initiation
- When the holoenzyme slides into a promoter it transitions to a closed binary complex
- If the sigma factor interacts strongly with the promoter, the holoenzyme transitions to an unstable open complex
- Several rNTPs are incorporated into the transcript, forming the ternary complex
- The RNAP transitions into a ternary elongation complex
a closed binary complex is
reversible
open complex
DNA duplex is bent 90° in order to place it into the active site
Promoter is denatured from -11 to +3 with assistance from sigma
Transcription bubble increases to 22-24 nt in length
Jaws close around downstream sequence
Transition to open state is irreversible
ternary complex
Additional rNTPs are added to the transcript while the RNAP remains tightly bound to the promoter
RNAP is not able to move down the template
RNAP pulls upstream DNA into the active site via a “scrunching” mechanism
Scrunching creates considerable stress that results in release of these short transcripts
Abortive initiation transcripts of 15-20 nucleotides
Energy of successive abortive initiation events is used to eventually break RNAP free from the promoter and transition to elongation
ternary elongation complex
Sigma factor is released
Bubble returns to 10-12 nt in length
RNAP coverts into the core enzyme
promoters are
cis-acting control elements
In a typical bacterial genome, a … bp sequence would be the minimum length required to have a unique recognition site
12
the sequence of the promotor does not need to be
contiguous
important conservation is localized in
short consensus sequences
promoter structure
- purine at transcription start site
- -10 element
- -35 element
- Space between the -10 and -35 elements
- base pairs between -10 and +1
- Extended -10 element
- UP element
the space between -10 and -35 elements
Is 16-18 bp in 90% of promoters
Sequence is unimportant
Determines the separation between interacting regions of RNA polymerase
Also determines the geometric orientation of DNA helix with respect to interactions with RNAP holoenzyme
base pairs between -10 and +1
Discriminator sequence
Base pairs downstream of -10 on the nontemplate strand
Interactions between these base pairs and 2 are important for stability of the open complex
extended -10 element
Upstream of the -10 element
TGN sequence at this position can compensate for a -35 element that does not closely match the -35 consensus sequence
UP element
20 bp region upstream of the -35 element
Interacts with the CTD
When the UP closely matches the consensus, transcription is greatly increased
down mutations
Usually reduce conformance to the consensus sequences
Decrease promoter efficiency
up mutations
increase conformance to the consensus sequence
increase promoter effiency
Mutations in the -35 sequence can affect
initial binding of RNA polymerase
Mutations in the -10 sequence can affect
Binding of RNA polymerase
The efficiency of the melting reaction that converts closed to open complex
Some promoters lack recognizable -10 and -35 elements and Require
ancillary activator proteins to recruit RNA polymerase and initiate transcription
Different sigma factors bind to the core polymerase in a similar way Even though
different sigma factors have different amino acid sequences
sigma 70
associated with basal gene expression
alpha helical domains of sigma 70 associate with
the promoter
N-terminal domain of sigma is
autoinhibitory
N-terminal domain of sigma functions
Normally masks the DNA binding domain of sigma
Prevents sigma from nonspecifically binding to and blocking promoters
Swings out of the way once sigma binds to the core RNAP
N-terminal domain also blocks the DNA-binding domain of the holoenzyme until an open complex is formed
The consensus sequences at -35 and -10 provide most of
the contact points for RNA polymerase in the promoter
a nonspecific interaction
occurs between sigma2 and only the phosphodiester backbone in the closed binary RNAP complex
a specific interaction
between sigma 2 and base pairs of the -10 and discriminator facilitates the melting that leads to the irreversible transition to the open RNAP complex
A portion of sigma must be displaced to accommodate RNA synthesis
The sigma3-sigma4 linker mimics RNA
Lies in the middle of the RNA exit channel
Must be ejected in order to produce a transcript > 20 nt in length
Loss of interaction between sigma3-sigma4 and RNAP is often associated with release of entire sigma and transition to core RNAP and elongation
The DNA unwound during scrunching is rewound
Energy of DNA rewinding is used to break -core RNAP and promoter-RNAP interactions
Scrunching allows for storage and use of energy needed to transition to elongation
All RNA polymerases share
similar structures and mechanisms
shared RNA polymerase structures and mechanisms
DNA-binding primary channel lined with positive charges
active site with Mg2+
ssDNA is bent at 90 degree angle
RNA polymerase moves forward using a
Brownian ratchet mechanism
Brownian motion leads to
random fluctuations of structures in the active site
Binding of the correct rNTP will stabilize the
active site structure in the active conformation
A “trigger loop” becomes … when a correct rNTP enters the active site
folded
Any event that displaces the 3’-OH of the RNA transcript from the active site will interrupt
core polymerase action
The core polymerase has an … function that is able to remove mispaired nucleotides in order to restore correct RNA placement and activity
exonuclease
The exonuclease function is stimulated by accessory factors
Accessory factors insert a narrow domain into the RNA polymerase
Domain closely approaches the active site
Allows a second Mg2+ to enter the active site
Converts active site from polymerase function to exonuclease function
The core RNA polymerase does not move at a constant rate
Can pause or backtrack
Movement determined by context of template strand sequence
at a true terminator
Extended pause
All hydrogen bonds of RNA-DNA hybrid are broken
DNA duplex reforms
extrinsic terminators
require Rho
Intrinsic terminators have two sequence features
G+C rich hairpin loop formation in RNA transcript
Up to seven uracil nucleotides in the RNA transcript following the hairpin region
the hairpin induces
a change in RNA polymerase activity that leads to a disruption in the active site and dissociation of U-A base pairs
readthrough transcripts
Transcripts that are not stopped by the terminator
Can be facilitated by factors that interact with RNA polymerase and/or the RNA transcript
Antitermination
Mutational studies have shown the importance of the
G-C base pairs of the hairpin and U-rich downstream region
Other upstream and downstream sequences also influence the
effiecency of termination
extrinsic termination
- Rho binds to rut sites in the RNA transcript upstream of the termination site
- Rho moves downstream along the RNA transcript
- The RNA polymerase will pause
- Rho invades the active site and uses its intrinsic helicase function to break the RNA-DNA hybrid
Rho
Hexameric ATP-dependent helicase
How does Rho work
RNA is wound from the 3’ end around the N-terminal RNA binding domains
5’ end of RNA pushed through interior of ring to ATP binding domains
ATP hydrolysis used to translocate Rho in a 3’ direction down RNA
heat shock response
is a conserved pathway that protects cells from temperature stress
Increased denaturing of proteins
products of heat shock response
Chaperones that refold proteins
Proteases that degrade denatured proteins
sigma factor for heat shock
sigma 32
Unfolded protein levels decrease after a heat shock response has activated
Concentration of unoccupied proteases and chaperones increases
Proteases degrade free sigma32 at a greater rate
Relative concentration of sigma32 decreases
Relative concentration of sigma70 increases
RNA polymerases are less likely to complex with sigma32
