Chapter 21: Transcription Flashcards
What is transcription?
DNA dependent synthesis of RNA
What is mRNA, tRNA, and rRNA?
mRNA: Encodes amino acid sequence of proteins (instructions)
tRNA: Reads info in the mRNA (adapter)
rRNA: Consitutes ribosome, makes proteins (machinery)
What are the functions of other RNAs?
Regulatory or catalytic function
Which strand is used to synthesize the mRNA strand?
The antisense strand (complimentary). The mRNA is identical to the sense strand. Thymine gets replaced with uracil in RNA.
What are the three steps in transcription?
Initiation, Elongation, Termination
Compare and contrast Transcription and replication?
Transcription:
- No primer needed
- Involves limited segments of DNA
- Only one DNA strand serves as the template
Replication
- Requires primer
- Involves entire strand of DNA
- Requires both strands of DNA
What does RNA polymerase do?
- Search for initiation (promoter)sites
- Unwind DNA to produce single strand DNA template
- Catalyze formation of a phosphodiester bond
- Detect terminal signals
- Interact with activator and repressor proteins that regulate transcription
How is proofreading in Transcription different from DNA replication?
- Higher error rate (1 in 10^4 to 10^5 ribonucleotides)
- Lack 3’ ->5’ proofreading exonuclease activity
- RNA Pol corrects errors by removing mismatch from 3’ end by reversing Pol reaction
- Lower RNA synthesis fidelity can be tolderated because it is not transmitted to progeny (except viruses)
- RNA eventually is degraded and replaced
What do the subunits of RNA Pol in E.coli do?
What does the sigma(σ) factor do?
Interacts with the core of RNA Pol to recognize the -35 and -10 boxes
What is a promoter?
- A sequence where RNA Pol can bind around boxes -35 and -10 which are important for σ70 subunit
How is the strength of a promoter sequence determined and what does that mean for the transcription rate?
- If a gene has a strong promoter which will match the consensus sequence
- Will be transcribed frequently (every two seconds)
- If a gene has a weak promoter it wont match the consensus sequence well
- Will be transcribed less frequently (every 10 minutes)
Draw the transcript bubble and include the channels and labeled ends.
Outline the steps of prokaryotic transcription.
- RNA Pol binds to promoter at -35 and -10
- DNA is partially unwound
- RNA synthesis initiated in a primer-independent reaction
- Sigma factor is released
- elongation continues (28-80 bases/s)
Describe the termination of Transcription.
- Termination signaled by specific sequences
- If RNA Pol is released prematurely it must start over again
- E.coli has two termination signals
- Rho dependent (Rho is a protein factor)
- Rho independent
Describe Rho-dependent termination.
- Caused by Rho binding to C rich region
- Rho is a helicase that destabilizes the RNA:DNA helix thus terminates transcription
Describe Rho-independent termination.
- Intrinsic termination
- Depends on presence of certain sequence
- Termination sequence on the template consist of GC rich followed by AT rich region
- Forms stem loop at GC rich region
- Stem loop causes Pol to pause and causes unstable buplex to dissociate
How is Bacterial transcription regulated?
- Polymerase binding and initiation are what is regulated
- Proteins can bind to promoter to affect gene expression
- Activator: CRP (cAMP repressor)
- Repressor: Lac
Generally describe eukaryotic transcription
- Extensive transcript modification
- Most mRNA encode single gene (monocistronic)
- Many accessory factors involved
- Transcription and translation are uncoupled
What are the RNA polymerases in eukaryotes and what do they do?
RNA Pol I: synthesize rRNAs
RNA Pol III: synthesize tRNA
RNA Pol II: responsible for synthesis of mRNA
What is a CTD?
- Carboxyl-terminal domain
- consists of many repeats of a consensus heptad aa sequence (YSPTSPS)
What is RNA Pol II important for in Eukaryotes?
RNA Pol II is central to protein coding genes
What is a transcription factor?
- Regulatory proteins that bind to promoters
- Eukaryotes have complex genomes thus need more proteins
- Certain transcription factors function as bacterial sigma factors and help recruit RNA Pol to promoters depending on growth conditions
Outline the regions involved in transcription (promoter related).
- TATA box
- Inr: initiator region/ start site
- Py n is n# of pyrimidines
- Promoter: Where TATA Binding Protein binds
- TBP analogous with sigma 70 in bacteria
Outline the steps involved in the loading of transcription factors to Pol II.
- TFIID and TBP bind to TATA
- TFIIA and TFIIB bind to complex
- TFIIE and TFIIH bind to complex followed by RNA Pol II with other factors
- Ribonucleotides are added as needed
What happens when the TFs have been loaded and RNA Pol II has attached?
- DNA unwound at Inr region
- CTD of Pol II is phosphorylated causing it to escape the promoter and start transcription
- Pol II enters elongation phase which releases transcription factors then is enhanced by elongation factors
What happens in termination
- Termination is facilitated by termination factors
- Elongation factors dissociate, CTD is dephosphorylated as transcription stops and RNA Pol II is released
What are some RNA Pol inhibitors?
Actinomycin D
- Inhibits elongation in Bact and Euk
- Prevents Pol movement
Rifampicin
- Binds to B subunit of Bact RNA Pol
- Prevents promoter clearance step
Alpha-Amanitin (from mushroom)
- Blocks Pol II and Pol III
- Doesnt block it’s or Bact’s Pol I
Describe the RNA processing in Eukaryotes
- Newly synthesized RNA molecule is called Primary transcript
- Primary transcript typically includes introns
- RNA splicing is when introns are removed and exons are joined together
What are the three steps to turn a primary transcript to mRNA?
- 5’ Cap is added to protect degredation and aid in translation start
- Poly-A tail is added to prolong RNA life
- Added by Poly-a Polymerase
- Requires a portion of 3’ to be removed
- Added by Poly-a Polymerase
- Introns removed requires snRNPs
What does the CTD of RNA Pol II do?
- Binds mRNA capping
- Splices
- Binds polyadenylation factors
- Terminates transcription
Why does splicing result in the formation of a lariat?
When Precursor mRNA is processed, splicing factors will remove an intron and join it with itself forming a lariat and joining the exons to form a mature mRNA
What does intron splicing look like?
What are the four types of introns?
Group I and Group II
- Self splicing
- GI found in nuclear mito, chloro genes coding for r/m/tRNA
- GII found in transcripts of mito or chrolo mRNA in fungi, plants, algae
Spliceosomal introns are spliced by splicosomes
- Most common
- Found in protein coding region of euk. genome
tRNA introns spliced by protein based enzymes
- Primary transcripts cleaved by endonuclease
- Exons are joined by ATP-dependent ligase
What happens to spliceosomal introns?
- Intron removal catalyzed by spliceosome
- Forms Lariat of intron and joins the exons
Describe the spliceosome.
- Composed of highly conservative RNA proteins (snRNPs)
- snRNP contain snRNAs (100-200 nucleotides long in nucl)
- snRNAs involved in splicing: U1, U2, U4, U5, U6
- GU at 5’ AG at 3’ mark sites of splicing
What is alternative processing?
When mRNA is processed in more than one way to make different
Draw the central dogma of DNA and RNA.
