Chapter 21: Transcription Flashcards by Rene Galindo

What is transcription?

DNA dependent synthesis of RNA

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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)

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What are the functions of other RNAs?

Regulatory or catalytic function

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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.

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What are the three steps in transcription?

Initiation, Elongation, Termination

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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

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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

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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

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What do the subunits of RNA Pol in E.coli do?

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What does the sigma(σ) factor do?

Interacts with the core of RNA Pol to recognize the -35 and -10 boxes

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What is a promoter?

A sequence where RNA Pol can bind around boxes -35 and -10 which are important for σ70 subunit

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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)

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Draw the transcript bubble and include the channels and labeled ends.

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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)

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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

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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 - 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.