Molecular Chapter 19
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What is the basic structure of the transcription bubble?
The structure formed by unparied DNA during transcription in which RNA polymerase uses one strand as a template to synthesize mRNA.
What is the function of the various subunits of eubacterial RNA polymerase?
- 1. Alpha - enzyme assembly, promoter recognition, activator binding
- 2. Beta/Beta' - catalytic center
- 3. Sigma - promoter recognition
- 4. Omega - enzyme assembly, regulation
Distinguish between holoenzyme and core enzyme.
- 1. Core enzyme binds indiscriminately to any DNA
- 2. Holoenzyme (w/sigma factor) has specificity for promoters
What are the stages of initiation and the complexes formed during these stages
- 1. Closed complex - The promoter is recognized
- 2. Open complex - A transcription bubble is created
- 3. Ternary complex - RNA polymerase bind to DNA to begin synthesis
Describe the key promoter consensus sequence elements.
- 1. Purine at the startpoint
- 2. A TATAAT hexamer centered at -10 (-10 element or TATA box)
- 3. A TTGACA hexamer centered at -35 (-35 element)
What is a down mutation?
A mutation in the promoter region that reduces transcription
What is an up mutation?
Mutations in the promoter region that enhance transcription
Why does sigma factor not bind DNA as a free subunit and what is the means by which different sigma factors are able to recognize different promoters?
- 1. In free sigma 70, the N-termnal region of sigma 70 mimics DNA and binds its own DNA binding domains
- 2. When sigma binds with core enzyme, the conformation changes, freeing the DNA binding domains to bind DNA specific promoter sequences
What is footprinting?
- An analysis technique to determine the protein binding site on DNA.
- 1. Control DNA is digested and produces fragments
- 2. Experimental DNA is bound with a protein that protects the binding region from digestion
- 3. Both are analyzed using gel electrophoresis
Distinguish between intrinsic termination and Rho factor dependent termination.
- 1. The terminator sequence codes for a G+C rich hairpin structure in the product followed by a series of up to 7 U residues.
- 2. The formation of the hairpin snaps that product from RNA Pol, releasing it from the DNA and terminating transcription
1. Rho factor binds to nacent RNA and tracks along the RNA to interact w/RNA pol and release it from the elongation complex
What is meant by polarity effect mutations?
- 1. WT - Ribosome on the nascent RNA strand prevents RNA pol access by RHO and transcription continues to end
- 2. Non-sense mutation - Ribosome falls off nascent strand due to mutation and RHO terminates transcription prematurely
Describe the twin domain model for transcription.
Transcription generates more tightly wound (positively supercoiled) DNA ahead of RNA polymerase, while the DNA behind becomes less tightly would (negatively supercoiled)
What is the role of sigma factor substitution?
- 1. The sigma factor associated with core enzyme determines the set of promoters at which transcription is initiated
- 2. Environmental conditions can induce a specific sigma factor to provide some adaptation to the environment
What is meant by cascade gene regulation?
- 1. The early genes are transcribed by host RNA pol
- 2. One of the early genes codes for a sigma factor that causes RNA pol to transcribe the middle genes
- 3. Two of the middle genes code for subunits of sigma factor that causes RNA pol to transcribe the late genes
What is antitermination?
An antitermination protein can act on RNA pol to enable it to read through a specific terminator.
Describe the bacterial mRNA life cycle.
- 0 min - Transcription begins
- 0.5 min - Ribosomes begin translation
- 1.5 min - Degradation begins at 5' end
- 2.0 min - RNA pol terminates at 3' end
- 3.0 min - Degradation continues, ribosomes complete translation
Describe the basic structure of a bacterial polycistronic mRNA.
5' UTR --> Start --> Coding region --> Stop --> Intercistronic region --> Start --> Coding region --> Stop --> 3' UTR
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