Lecture 45- Prokaryotic Transcription & Gene Control Flashcards
What are trans-acting factors?
Examples Include:
diffusible and thus can function at multiple sites in genome
DNA binding protein such as transcription factors, some regulatory RNAs
What are cis acting elements?
Examples Include:
tied closely to the gene
DNA sequence elements such as promoters and binding sites for trans-acting factors
What is RNA and what are it’s 4 defining characteristics?
linear single-stranded polynucleotide chain with:
- sequence always read 5’–>3’
- ribose-phosphate backbond
- U instead of T
- intramolecular base pairing that yields complex secondary and tertiary structure
Define messenger RNA (mRNA)
RNA copy of coding strand of DNA from bacterial gene. Carries base sequence encoding amino acid sequence for a protein.
Define transfer RNA (tRNA)
RNA molecule that carries AA into catalytic site of ribosome. tRNA base pairs to mRNA to ensure selection of correct AA to be incorporated into nascent polypeptide chain
Define ribosomal RNA (rRNA)
Structural components of ribosome, enzyme that catalyzes protein synthesis
What is a transcription unit and the process of transcription?
DNA that can be transcribed into a single RNA molecule catalyed by RNA polymerase
Where does a transcription unit start and end?
What is the finished molecule?
stars at promoter, ends at terminator
Primary transcript
During mRNA synthesis, what is considered upstream and downstream?
Upstream: closer to the 5’ end
Downstream: closer to the 3’ end
What gives the location of the promoter and what is its value?
Transcription start site, +1
What term defines close to the promoter?
Far from the promoter?
Proximal
Distal
The RNA __ sequence is similar to the DNA coding strand
The RNA TRANSCRIPT sequence is similar to the DNA coding strand
Original 2 stranded DNA consists of?
Coding and template strand
When DNA gets unwound, we use the ____ strand to transcribe. The ____ that is formed is the compliment of the ____ strand.
When DNA gets unwound, we use the template strand to transcribe. The transcript that is formed is the compliment of the template strand.
How are coding and template strands defined?
For each individual gene
What are operons?
coordinately regulated gene clusters, cluster of bacterial genes
___ are arranged in 5’ –> 3’ in transcription unit
OPEN READING FRAMES are arranged in 5’ –> 3’ in transcription unit
Use of one promoter and terminator yields a ____
Use of one promoter and terminator yields a POLYCISTRONIC mRNA
How many proteins does the atp operon encode?
9
A ___ is a cis-acting element in a genome where ___ ______ binds to initiate transcription
A PROMOTER is a cis-acting element in a genome where RNA POLYMERASE binds to initiate transcription
Where are bacterial promoters’ defined sequence elements?
- the -35 region
- the -10 region
Where is the spacer in prokaryotic promoters?
between -35 and -10 regions
What do most RNA transcripts start with?
Purine (+1) within the sequence CAT
What is the bacterial consenus promoter?
-35 Spacer -10 Spacer +1
TTGACA– 16-18bp – TATAAT –5-9bp - CAT
What are constitutive promoters?
Actively initiate transcription continuously for ‘housekeeping genes’
What does the constitutive promoter strength depend on?
sequence
What is a major determinant of the rate of transcription initiation for constitutive promoter?
Similarity to consensus sequence
The closer to the consensus sequence the promoter is, the faster or slower the rate of transcription?
faster
How many polymerases does E.coli have that catalyzes transcription in 5’ –>3’. What is it (they) called?
1, but 2 types
RNA polymerase (465kD)
What does RNA polymerase holoenzyme do?
Responsible for transcription initiation and synthesis of first 10 nucleotides of RNA chain
What are the subunits of the holoenzyme?
alpha2, beta, beta’, omega, sigma,
- sigma recognizes promoter
- alpha2 (40kd) essentail for enzyme assembly involved in interaction with activators
- beta (155kD) and beta’ (160kD) form catalytic core
- omega provides structural stability
What does RNA polymerase core enzyme consist of and do?
alpha2, beta, beta’, omega
carries out transcription elongation
What holoenzyme binds the most promoters in the E.coli genome and what is its function?
sigma 70! (70% are sigma70)
housekeeping
What does RNA polymerase require (3)?
- DNA template
- no primer
- substrates that are ribonucleotide triphosphates
T/F: RNA polymerases have proof-reading exonuclease activity.
Why/why not?
FALSE. RNA polymerases DO NOT have proof-reading exonuclease activity. (Error rate: 10-4 to 10-5)
It can afford it bc defective mRNA –> protein which will undergo turnover quickly. Whereas, if DNA makes a mistake, mutation in the genome could kill the cell
Transcription Initiation
- __ subunit binds to the ______
- Rest of the RNA pol binds to the ____ subunit which produces a _______ that binds to the promoter and creates a “_____” complex
- Unwinding of DNA forms a ______ _____, #-#bp, to produce an “___” complex
- ________ initiates RNA synthesis for ~__ nt
Transcription Initiation
- Sigma subunit binds to the promoter
- Rest of the RNA pol binds to the Sigma subunit which produces a holoenzyme that binds to the promoter and creates a “closed” complex
- Unwinding of DNA forms a transcription bubble, 12-15 bp, to produce an “open” complex
- Holoenzyme initiates RNA synthesis for ~10 nt
Transcription Elongation
- Dissociation of the __ factor produces the _____ enzyme. “______ ______”
- ____ replaces ___ subunit
- Transcription elongation by _____ enzyme ~#-# bases/sec, but slowed by formation of ______ _____
- Transcription ______: release of ____ and dissociation of _____ enzyme from ____
Transcription Elongation
- Dissociation of the sigma factor “Promoter Clearage” produces the core enzyme.
- NusA replaces sigma subunit
- Transcription elongation by core enzyme ~50-90 bases/sec, but slowed by formation of secondary structure
- Transcription termination: release of RNA and dissociation of core enzyme from DNA
