Week 28 / Transcription Flashcards
Q: What is transcription?
[what is it ?
what does it form?
]
A: Transcription is the enzymatic synthesis of RNA from a DNA template and
forms the first step in gene expression.
Q: What is the product of transcription?
A: The product of transcription is messenger RNA (mRNA).
Q: What is translation?
A: Translation is the enzymatic synthesis of protein from a transcribed gene sequence into a functional RNA molecule (mRNA).
Q: What enzyme catalyzes transcription?
A: Transcription is catalyzed by an RNA polymerase enzyme complex.
Q: What are the requirements for transcription? [2]
A: Transcription requires
a dsDNA template and ribonucleotides (ATP, GTP, CTP, and UTP).
Q: In which direction does RNA synthesis occur?
A: RNA synthesis occurs in the 5’ to 3’ direction.
Q: What are the two DNA strands used in transcription?
A:
Sense strand: Carries the gene sequence that will be copied into an RNA molecule for protein translation.
Antisense strand: Used as a template to generate a complementary RNA copy and also carries sequences for non-coding RNA molecules with structural or regulatory functions.
Q: How many RNA polymerases are present in prokaryotes?
A: Prokaryotes have a single RNA polymerase.
Q: How many RNA polymerases are present in eukaryotes?
A: Eukaryotes have three RNA polymerases: I, II, and III.
Q: What is the function of RNA Polymerase II in eukaryotes?
A: RNA Polymerase II transcribes all protein-coding genes.
Q: What is the function of RNA Polymerase I in eukaryotes?
A: RNA Polymerase I transcribes most ribosomal RNAs (rRNAs).
Q: What is the function of RNA Polymerase III in eukaryotes?
A: RNA Polymerase III transcribes transfer RNAs (tRNAs).
Q: What are the steps involved in transcription initiation? [5]
A:
RNA Polymerase Binding – RNA polymerase binds to the dsDNA.
Promoter Recognition – The polymerase binds to a promoter sequence upstream of the gene.
Promoter Strength & Regulation – Promoters can be weak or strong depending on sequence elements and protein stimulation.
DNA Unwinding – The dsDNA is locally unwound so RNA polymerase can access the template strand.
Start Site Selection – RNA polymerase begins RNA synthesis at the start site (position +1) of the gene.
Q: What are the steps involved in transcription elongation? [4]
A:
Nucleotide Addition – RNA polymerase covalently adds ribonucleotides to the 3’-end of the growing mRNA molecule in the 5’ to 3’ direction.
Template Strand Reading – The polymerase moves along the antisense/template DNA strand in the 3’ to 5’ direction.
DNA Unwinding & Rewinding – The polymerase locally unwinds the DNA ahead and reforms the helix behind it.
Elongation Speed – In E. coli, RNA polymerase synthesizes around 40 bases per second at 37°C.
Q: How many subunits are there in E. coli RNA polymerase?
A: E. coli RNA polymerase consists of at least five known subunits.
Q: What is the role of the α-subunits in E. coli RNA polymerase?
A: The two α-subunits are required for holoenzyme assembly.
Q: What does the β-subunit do in E. coli RNA polymerase?
A: The β-subunit is the catalytic center of the enzyme and is key for initiation and elongation.
Q: What is the function of the β’-subunit in E. coli RNA polymerase?
A: The β’-subunit binds two Zn²⁺ ions to help catalyze the joining of ribonucleotides.
Q: What is the function of the σ-subunit in E. coli RNA polymerase?
A: The σ-subunit is responsible for promoter recognition.
Q: What is the role of the ω-subunit in E. coli RNA polymerase?
A: The ω-subunit stabilizes the assembled holoenzyme.
Q: What determines which promoter is recognized by E. coli RNA polymerase?
A: Different σ factors/subunits recognize different promoters. The most common σ factor in E. coli is the σ⁷⁰ factor.
Q: What does the E. coli RNA polymerase bind to in the promoter region?
A: The E. coli RNA polymerase binds to sequences within the promoter region upstream of the initiation site.
Q: How is the transcription start site and promoter sequence numbered in E. coli?
A: The transcription start site is denoted by position +1, while the promoter sequence is denoted by a negative number since it is upstream of the initiation site.
Q: What is the length of the σ⁷⁰ promoter in E. coli?
A: The σ⁷⁰ promoter is between 40 and 60 base pairs long.
Q: Which regions of the σ⁷⁰ promoter are associated with RNA polymerase holoenzyme binding?
A: The region from -55 to +20 binds the RNA polymerase holoenzyme, and the region from -20 to +20 is very strongly associated with the holoenzyme.
Q: What is required for efficient transcription in E. coli?
A: Sequences up to -40 are required for efficient transcription.
Q: What happens during Step 1 (Promoter Binding) of E. coli transcription?
Core Polymerase Alone:
Has loose, non-specific DNA binding.
σ Factor Joins → Holoenzyme:
Forms the holoenzyme.
Gains 100x stronger affinity for specific promoter regions.
Promoter Search:
Holoenzyme slides along DNA, looking for -35 and -10 promoter sites.
Closed Complex Formation:
Binds to promoter but DNA is still closed.
Waits for signals at important genes to begin transcription.
A:
The core polymerase enzyme has a loose, non-specific affinity for DNA.
Association of the σ factor forms the holoenzyme, which binds to specific promoter sequences with a 100-fold increase in affinity.
The holoenzyme is thought to slide along the DNA, searching for the promoter’s -35 and -10 sites.
This closed complex awaits stimulation at highly expressed or important genes.
Q: What happens during Step 2 (DNA Unwinding) of E. coli transcription?
Polymerase Unwinds DNA:
To access the antisense strand (the template for transcription).
DNA Topology Helps:
Negative supercoiling at active promoters makes DNA easier to unwind.
Promoter Site Positioning:
The -35 and -10 sites are spaced to support DNA bending/unwinding.
Open Complex Forms:
Unwinding leads to an open complex with the holoenzyme bound to single-stranded DNA.
A:
The polymerase unwinds the DNA to access the antisense strand.
The DNA topology plays a key role, with negative supercoiling used at active gene promoters to help melt the duplex.
The -35 and -10 sites’ relative positions facilitate DNA conformation changes.
The initial unwinding forms an open complex with the holoenzyme.
Q: What occurs in Step 3 (Transcription Initiation) of E. coli transcription?
No Primer Needed:
RNA synthesis starts without a primer (unlike DNA).
First Bond Formation:
Begins with GTP or ATP, forming a phosphodiester bond between the first 2 ribonucleotides.
Abortive Initiation:
First 9 bases are added while the enzyme is stationary—may abort and retry multiple times.
Speed Matters:
Especially during rapid growth, transcription decisions must be quick and efficient.
A:
RNA synthesis begins without a primer (unlike DNA synthesis).
The first two ribonucleotides are incorporated to form a phosphodiester bond, starting with GTP or ATP.
The first 9 bases are joined without the enzyme moving; transcription can be aborted during this phase.
The process is fast, especially during rapid growth, requiring quick decision-making for transcription initiation.
Q: What happens during Step 4 (mRNA Elongation) of E. coli transcription?
Ternary Complex Forms:
σ-factor + Polymerase + DNA + nascent RNA → forms a ternary complex.
This triggers polymerase movement and promoter clearance.
Elongation Begins:
Polymerase moves at ~40 bases/sec.
Keeps 17 base pairs unwound and maintains a 12 bp RNA-DNA hybrid.
σ-factor Release:
Once the polymerase starts elongation, the σ-factor is released.
It’s free to join another core polymerase and repeat the process.
A:
The σ-factor forms a ternary complex with polymerase, DNA, and nascent RNA, causing the polymerase to move forward and clear the promoter.
The polymerase moves at 40 bases per second, keeping 17 base pairs unwound and a 12 base pair RNA-DNA hybrid constant.
The σ-factor is released once the polymerase is moving, and it is free to form a new holoenzyme.
Q: What occurs during Step 5 (Termination) of E. coli transcription?
Termination Signal Reached:
RNA polymerase stops when it hits a termination sequence.
Hairpin Structure Forms:
RNA folds into a GC-rich hairpin using self-complementary regions.
This hairpin is very stable and stalls the polymerase.
U-Rich Sequence Follows:
After the hairpin, RNA has 4+ uridine (U) bases.
These weakly bind to the A bases on the DNA template.
Dissociation:
The weak U-A bonds + stalled polymerase cause the enzyme to fall off the DNA.
A:
The polymerase dissociates from the DNA upon reaching a termination sequence.
The common stop signal is an RNA hairpin formed by self-complementary sequence areas in the mRNA.
The hairpin is GC-rich and stable, stalling the polymerase.
After the hairpin, a sequence of 4 or more uridine (U) residues weakly binds to the adenine (A) residues on the template strand, causing dissociation of the core enzyme.
Q: How is transcription controlled in most genes?
A: Transcription of a particular gene is controlled by a regulatory region of DNA near the site of transcription. This is common in prokaryotes.
Q: What are the two types of regulatory regions in transcription control?
A:
Some regulatory regions act like simple switches.
Others act like a microprocessor, responding to a variety of signal inputs, which is more common in eukaryotes.
Q: How do gene regulatory proteins control gene transcription?
A: Gene regulatory proteins bind to the regulatory region of the DNA to turn genes on or off.
Q: How do gene regulatory proteins interact with DNA?
A: The proteins bind to the outside of the DNA by recognizing specific sequences, interacting with the exposed edges of base pairs via hydrogen bonding and hydrophobic interactions in both the minor and major grooves.
Q: Where do most gene regulatory proteins bind on the DNA?
A: Most regulatory proteins bind to the major groove of the DNA because the patterns for each of the 4 base pairs are unique there.
Q: How long are the regulatory regions of DNA that gene regulatory proteins bind to?
A: These regulatory regions typically contain less than 20 nucleotide base pairs.
Q: What is the role of Zn²⁺ ions in some DNA-binding proteins?
A: Some DNA-binding proteins use Zn²⁺ ions to help maintain the correct protein folding.
