RNA Section Flashcards
1
Q
RNA Polymerase Structure
A
- Total of five subunits consisting of two identical alpha subunits, two different betta subunit, and a sigma subunit
- Low affinity holoenzyme consist of all 5 subunit, identifies DNA to be transcribed
- Recognition helix of sigma subunit performs identification
- Once promoter region identified, sigma subunit dissociates leaving high affinity core enzyme
- Core enzyme has active site to facilitate RNA synthesis
- Prokaryotes have 5 subunits, eukaryotes have 7-10 subunits
2
Q
Core enzyme active site
A
- Active site similar structure to DNA polymerase
- Magensium ion in active site coordinated to three key aspartic acid residues (D256, 192, 190) and RNA transcript
- When ribonucleotide triphosphate enters active site, it brings second magnesium ion
- Same active site in both eukaryotes and prokaryotes
3
Q
RNA polymerase function
A
- Recognition of initiation sites: For prokaryote, sigma subunit identify initiation site by recognition helix. For eukaryote, use recognition helix of TATA binding protein
- Helicase activity: RNA polymerase can unwind dsDNA to ssDNA to form transcription bubble
- RNA synthesis: Connects nucleotides unidirectionally through formation of phosphodiester bonds
- Recognition of termination signals: Prokaryotes use intrinsic or protein mediated signals to stop
- Interaction with transcription factor: Interact with activating and inhibitory transcription factors to alter way it binds to DNA
- Occurs in 5’-3’ direction (DNA strand must be in 3’-5’)
- 3’-OH attacks 5’alpha phosphate of incoming nucleotide of incoming NTP -> release pyrophosphate and magnesium in active site. Pyrophosphate hydrolyzed to generate energy
4
Q
RNA vs DNA polymerase nucleic acid synthesis
A
- RNA polymerase doesn’t require a primer, relies solely on promoter region
- RNA polymerase lacks proofreading, high error rate
- RNA polymerase much slower transcribing at 50 nt/s, where DNA polymerase transcribe 800 nt/s
5
Q
Transcription terminology
A
- Template strand/antisense strand: strand of DNA being transcribed, read 3’-5’
- Non template strand/sense or coding strand: DNA strand that shares same nucleic acid sequence as RNA transcript, except with substitution of thymine with uracil
- Transcription site denoted as +1, nucleotide with negative number is upstream of initiation site (promoter sequences that recruit RNA polymerase). Nucleotide with positive number is downstream and included in RNA transcript
6
Q
Mechanism of promoter recognition in prokaryotes
A
- Relies on interaction between promoter region and recognition helix
- RNA polymerase holoenzyme scans for promoter regions, facilitated by amino acids in recognition helix
- Functional groups form hydrogen bonds with nucleotides that indicate presence of promoter region
7
Q
Footprinting experiments
A
- Reveals specific DNA binding sites of protein
- PCR amplification: DNA template strand is amplified and radiolabeled at 5’ end
- Set reaction conditions: one with RNA polymerase and one without
- Cleave samples with DNAse
- Gel electrophoresis to visualize possible cleavage sites in DNA
- For reaction without RNAP, a nick made at every site along DNA fragment
- For reaction with RNAP, DNA regions bounded by RNAP are protected from DNAse. Specific region on gel devoid of bands indicating promoter region
8
Q
Highly conserved promoters
A
- Two specific regions that consistently show resistance to cleavage
- Pribnow box (-10): characterized by highly conserved TATAAT
- Second region (-35): Considered a consensus sequence, most commonly observed nucleotides at each position within group of aligned sequences
- Regions spaced 11-15 base pairs apart on DNA strand (constitute 40 nucleotide core promoter region)
9
Q
Promoter strength
A
- Strong promoters contain -10 and -35 regions that match consensus sequences
- Have two regions spaced 17 nucleotides apart
- Genes with strong promoters are termed housekeeping genes
- Weak promoters deviate from ideal spacing between -10 and -35 regions
- Strength can be altered by regulatory proteins (can inhibit or activate transcription by binding near or to promoter region)
- UP elements increase promoter strength: 40-60 nuceotides upsteam of core and characterized by A/T region that can bind to alpha subunit of RNAP -> increase stability and increase transcription rate
10
Q
Alternative promoter sequences
A
- Specific environmental conditions can alter gene expression -> adaption using different sigma units that recognize alternative promoter sequences
- sigma32 synthesized from high heat conditions and associated with heat shock proteins -> prevent denaturation
- sigma54 synthesized under nitrogen low conditions promoting genes that use alternate energy sources
11
Q
Mechanism of transcription initiation
A
- Pentameric holoenzymes forms H-bonds with bases of DNA using recognition helix of sigma subunit
- Once promoter region is identified, pauses and forms closed promoter complex (transcription still reversible)
- RNA polymerase unwinds DNA and forms open promoter complex (transcription irreversible). Forms transcription bubble -> DNA segment usually A/T rich (less hydrogen bond mean easier to unwind)
- Holoenzyme incorporates a purine in catalytic site to initiate transcription. Pentamer add up to 10 nucleotide in RNA chain
- Once 10 nucleotides synthesized, sigma subunit is released and core enzyme bind to DNA -> elongation phase
12
Q
Movement of transcription bubble in elongation
A
- RNA polymerase unwind DNA at front of enzyme. At back of enzyme, DNA that has been transcribed is rewound back to double strand form.
- As RNA synthesized, 8 base pair DNA-RNA hybrid helix forms between DNA template and RNA transcript. Extends from RNA active site to 3’ end
- For RNA to exit transcription bubble RNA-DNA hybrid helix must be separated, facilitated by phenylalanine residue in exit channel
13
Q
Nucleotide addition cycle
A
- Open trigger loop allows incoming NTP to enter pre-insertion site of RNA polymerase
- Undergoes conformational change to form closed trigger loop, moves NTP from pre insertion site to actual insertion site. NTP is catalytically incorporated into RNA chain releasing pyrophosphate
- DNA translocated (shifting one base towards 5’ end). Translocation begin by transitioning trigger loop from closed to open. Wedged conformation with intermediate DNA
- Streptolydigin inhibits catalytic incorporation of nucleotide by binding to RNA polymerase active site
14
Q
Intrinsic termination in prokaryotes
A
- Specific nucleotide sequences can generate a stem loop structure
- G-C inverted repeats: base pair with each other to form stem portion of stem loop
- U-loop: Segment of uracils between two G-C repeats forms loop
- U-run: 6-8 uracils transcribed -> facilitate weaker H-bond interaction between DNA/RNA hybrid helix -> RNA polymerase falls off and release synthesized RNA product
15
Q
Protein mediated termination
A
- Rho factor plays role in synthesis of various sized rRNA species
- Prokaryotic DNA encodes 10S, 13S, 17S, and 23S rRNA, without Rho factor -> intrinsic termination at end of rRNA and make 23S rRNA
- If Rho present, bind to specific Rho termination site -> protein-mediated termination, lead to synthesis of smaller rRNA 10S, 13S, 17S
- Specific size depends on time point Rho factor added
- If at beginning, shortest 10S transcribed
16
Q
Binding of rho
A
- Rho binds to C rich site on nascent transcript called rut site
- Uses ATP hydrolysis to chase RNA polymerase
- When RNA polymerase reach G-C rich region called termination region, stalls and allow Rho to catch up
- When Rho reaches RNAP, uses ATP hydrolysis to unwind DNA-RNA hybrid
17
Q
Drugs inhibiting prokaryotic transcription
A
- Rifampicin: Binds to conserved channel that accomodates RNA/DNA hybrid. Prevents formation of initiation complex. Only effective if it binds before completion of transcription. Rifampicin exclusively found in prokaryotes
- Actinomycin: DNA intercalator that inserts itself dsDNA template and distort helix. Inefficient template for RNAP. Effective in both eukaryotes and prokaryotes. High doses used for cancer treatments
- Amanitin: alpha-Amanitin blocks transcription at two step during nucleotide addition cycle. Block open trigger loop from closing, prevent NTP from moving into insertion site. Block wedged trigger loop from reopening, preventing RNAP from accepting new NTP. Present in both eukaryotes and prokaryotes
18
Q
Post-transcriptional modification
A
- Used for synthesis of rRNA and tRNA
- One primary transcript that encodes 16S rRNA, 23S rRNA, 5S rRNA, and tRNA
- RNAse P and D cleave at 5’ and 3’ end to generate tRNA
- RNAse III cleave out all rRNA segment from primary transcript
- Endonucleases M16, M5, M23 trim rRNA transcripts to exact size
- Each component processed by nucleotide modification to confer specific function
19
Q
Differences between eukaryotic and prokaryotic transcription
A
- Prokaryotes: lack of nuclear membrane does not separate transcription and translation. Only tRNA and rRNA modified. One RNAP with 5 subunits
- Eukaryotes: Separation between transcription and translation. extensive processing, mRNA modified. Three RNAPs with 7 to 10 subunits
20
Q
Eukaryotic RNA Polymerases
A
- Type 1: Nucleous. 18S, 5.8S, 28S rRNA transcripts. Low binding affinity to aminitin. Recognizes A rich upstream promoter element (UPE), ribosomal initiation element
- Type 2: Nucleoplasm. pre-mRNA and snRNA transcripts. High binding affinity to aminitin. Recognizes TATA box, initiator element, downstream promoter element.
- ## Type 3: Nucleoplasm. tRNA, 5S rRNA transcripts. Intermediate binding affinity to aminitin. Recognizes downstream promoters
21
Q
Transcription initiation in eukaryotes
A
- Regulated through interaction with upstream enhancers located 1kbp away from promoter region
- Initiation element: Can be combine with TATA box or downstream promoter element
- Upstream promoter elements (UPE) enhance binding of RNAP. Include CAAT box and GC box -> provide supplementary binding sites for RBP3 subunit of RNAP II
- Transcriptional activity regulated through C terminal domain(CTD) of RPB1 subunit. Contains repeats of amino acids tyrosine, serine, proline, and threonine. Ser and Thr can be phosphorylated to modulate activity
- Upstream enhancers can be bound to activators or repressors. Long range protein-protein interactions done by DNA looping.
22
Q
Mechanism of transcription initiation
A
- Eukaryotic RNAP II doesnt have sigma subunit, use transcription factors
- TFIID recognizes TATA box binding protein (TBP) that contains recognition helix
- TFIIA stabilizes TBP
- TFIIF brings RNAP to TFIID bound TBP
- TFIIB, TFIIE, TFIIH also recruited to form basal transcription apparatus/pre initiation complex
- TFIIH unwinds DNA and phosphorylated CTD
- ALL TFs dissociate except TFIIF, RNAP enters elongation phase
