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107 Cards in this Set

  • Front
  • Back
What is needed for RNA transcription?
-DNA template
-Ribonucleoside 5'-triphosphates
-RNA polymerase
What is not needed for RNA transcription?
A Primer
What are the 3 major types of RNA?
mRNA - 5%
tRNA - 15%
rRNA - 80%
What are 2 other nonmajor types of RNA?
siRNA (small interfering)
microRNA
What do microRNA's do?
Bind mRNA to inhibit translation.
What is snRNA? What does it do?
Small nuclear RNA; it guides the slicing of the RNA endproduct.
What key molecule is necessary for transcription?
RNA Polymerase - it makes all 3 types.
What are 2 forms of RNA polymerase can exist?
-Core enzyme

-Holoenzyme
What is the RNA Pol core enzyme composed of?
2 alpha
1 Beta
1 Beta'
1 w
What makes the holoenzyme of RNA Pol?
The addition of a sigma unit.
What is the core enzyme itself responsible for?
Polymerization of the RNA molecule.
Can the core enzyme operate by itself?
No; without the sigma factor RNA Pol cannot recognize the promotor on the DNA template.
What KIND of molecule is RNA polymerase?
a Zn2+ containing metalloenzyme.
what kind of linkage is made during RNA polymerization?
The 3' OH of the RNA backbone nucleophilicly attacks the alpha phosphate of the incoming nucleotide.
In what direction is RNA synthesized?
5' to 3'.
What is a major difference in the error rate of transcription vs replication??
RNA polymerase has no proofreading capability in the 3'-5' direction so the error rate is higher.
What is the coding strand?
The nontemplate strand of DNA that can be used to decipher the primary AA sequence of the encoded protein.
What are the 3 stages of RNA synthesis?
1. Initiation
2. Elongation
3. Termination
What 3 things constitue an operon?
1. Promoter
2. Operator
3. Structural genes
What is an operon?
A genetic unit consisting of genes, and regulation regions.
What is the lac operon?
The genetic unit responsible for producing the enzymes that allow bacteria to utilize lactose.
What takes place in the initiation phase of bacterial transcription?
-RNA Polymerase holoenzyme binds the DNA template
-Scans DNA for promotor at -35
-Forms closed complex at -35
-Moves to 2nd site at -10
-DNA unwinds to form open complex at -10
-mRNA forms, sigma subunit is released
What kind of message is transcribed from the lac operon?
a polycistronic message - three distinct gene products are produced.
Where is the control region for the lac operon?
Between the LacI and LacZ genes
What is the difference between the closed and open complexes?
Closed: RNA pol is stalled at the promotor region, but the DNA helix is still together.
Open: DNA melts apart and polymerization can occur.
What is the difference betweenthe -35 and -10 regions of the DNA template?
At -35, the RNA polymerase holoenzyme simply binds to form the closed complex.
At -10, the DNA helix melts to form the open complex.
Why is the transition from closed to open complex the key event in DNA transcription?
Because it exposes the start sequence on the DNA template strand intended for transcription.
How is it that the DNA melts to facilitate formation of the open complex?
Bacterial DNA is normally negatively supercoiled, so the unwinding process is favorable.
What nucleotide do RNA chains start with?
ppp-A or ppp-G; a triphosphate purine nucleotide.
What happens to the sigma subunit after transcription initiation?
It gets released as the polymerase moves on to the rest of RNA synthesis.
Why is the sigma subunit so good?
Because it reduces nonspecific binding of RNA Pol to DNA by a factor of 10^4 -> hence transcription only occurs at the correct promotor regions.
What does the control regionof the lac operon consits of?
CAP
Promotor
Operator
What is CAP?
Cyclic AMP Binding Protein
What is consensus?
The most common nucleotide at a specific sequence in genes from several different organisms. (e.g., -10 region)
What is the consensus sequence at the -10 region in prokaryotic DNA?
TATAAT
Why aren't all -10 regions identical?
Because there are different sigma subunits that are specific for each different -10 region.
What is the result of deviation in consensus?
Variation in affinity of the sigma factor for operator sequences; allows for different specificities.
Why is it good for bacteria to have a variety of sigma factors?
Their levels vary in response to environmental changes to help the bugs adapt.
What are 3 examples of promoters recognized by different sigma subunits?
Standard - TATAAT
Heat-shock - CCCATNT
Nitrogen-starvation - TTGCA
How exactly does the sigma subunit change RNA Pol's interaction with promoters?
-Increases specificity
-Decreases affinity
What would make a STRONG promoter? What would be the result?
-Close conformity to the consensus sequence
-Result in frequent initiation
What happens during the Elongation phase of transcription?
-Elongation proceeds 5'->3'
-Energy comes from phosphate cleavage of incoming nucleotide and PPi hydrolysis.
-Multiple RNA Pol's sequentially bind promoter for continuous transcription.
When does the sigma factor dissociate?
Immediately after elongation begins.
What is the rate of elongation in E.coli?
App. 30 nt/sec
What facilitates formation of a transcription bubble?
-Negative superhelicity in the DNA template.
What does unwinding generate in the DNA being transcribed?
Torsional stress from:
-Pos supercoils ahead of the bubble
-Neg supercoils in its wake
How is torsional stress from transcription relieved?
By topoisomerases
What 2 antibiotics target topoisomerase II? What class are these drugs in?
-Norfloxacin
-Ciprofloxacin
Quinolone class
What are 2 drugs that interact with HUMAN topoisomerase?
What are they used to treat?
-Doxorubicin
-Etoposide
-Used for treating cancer
How do the topoisomerase-targeting drugs work?
They ENHANCE the cleaving of DNA-double strands so much that it overwhelms the cell's ability to repair the damage.
What antibiotic is used as an inhibitor of topoisomerases?
Novobiocin
How much DNA is unwound by each RNA polymerase molecule?
17 nt per polymerase molecule.
What are the 2 types of transcription termination that can occur?
1. Protein dependent

2. Protein independent
What is the protein referred in "protein-dependent termination?"
Rho
What is a similar feature of both termination alternatives?
Both depend on a hairpin loop structure.
What is the Rho protein?
An ATP-dependent helicase that binds nascent RNA, uses ATP energy to catch up to RNA Pol, and truncates the DNA/RNA helix.
WHat helps the Rho protein catch up to the highly processive polymerase?
-Txn bubble pauses as it hits a hairpin loop encoded in the DNA template.
Where is the active signal for txn termination then?
In the nascent RNA chain ..?
What is the more common mechanism for Txn termination?
The protein INDEPENDENT mechanism.
What is a palindrome?
A sequence of units (such as a strand of DNA) that has the property of reading the same in either direction
How does a palindrome aid in transcription termination?
As RNA is transcribed, 2 palindromic sequences are incorporated so that enough nt's are between them to allow a hairpin turn to form.
What role does the hairpin turn play in protein-independent termination?
It slows down RNA polymerase and causes it to pause.
What happens when RNA polymerase pauses?
The sequence of U's in the new RNA transcript that are bonded to complementary A's in the DNA template dissociate b/c the A-U bond is the weakest type possible.
So protein independent termination is caused by:
-Stable hairpin turn
-Pausing of DNAP
-Dissociation of weak AU bonds
What stabilizes the RNA hairpin turn?
It is rich in G-C, which are strong bonds.
What is a-amanitin?
a mushroom toxin directed at RNA polymerases in humans (euks)
How many RNA polymerases are found in eukaryotic cells?
3
What does RNA pol type I synthesize?
Where is it found in cells?
Ribosomal RNA - 18S, 28S, and 5.8S b/c it's in euks.
-Found in the nucleolus
What does RNA pol type II synthesize?
Where is it found in cells?
mRNA precursors (immature)
snRNA
-Found in the nucleoplasm
What does RNA pol type III synthesize?
Where is it found in cells?
This mkes tRNA and 5S rRNA
-Found in the nucleoplasm
Which RNA pol types are inhibited by a-amanitin?
How strongly?
II (strongly) and III (but only in high concentrations)
So list 5 inhibitors of transcription:
RADDC
-Rifampin
-a-amanitin
-Daunorubicin
-DActinomycin
-Cordycepin
What is rifampin's mechanism of inhibiting transcription? What disease is it usually for?
Binds the B-subunit of bacterial RNA Polymerase to inhibit txn initiation.
-Used to treat tuberculosis.
What is the mechanism of a-aminitin?
It is a potent inhibitor of RNA Polymerase II, the class that makes mRNA and snRNA.
What is the mechanism of Dactinomycin?
Intercalates between two G-C base pairs in DNA
What is the mechanism of Daunorubicin?
Intercalates between base pairs
What is the mechanism of cordycepin?
It lacks 3' OH so terminates the chain.
What is the major difference in txn between Proks/Euks in terms of space/time?
Txn is compartmentalized in Euks -> the RNA transcript must be transported from the nucleus before translation can begin. Poses a point for regulation.
What is the eukaryotic promoter region?
the TATA Box
Where is the TATA box located?
-30 upstream
What prokaryotic sequence is the TATA box analogous to?
The pribnow box (-10)
What is the mechanism of Daunorubicin?
Intercalates between base pairs
What eukaryotic sequence is analogous to the prok -35 promoter sequence?
The -80 CCAT sequence.
What is the mechanism of cordycepin?
It lacks 3' OH so terminates the chain.
So what is the eukaryotic promoter region made up of?
Two consensus regions:
-80 CCAT
-30 TATA
What is the major difference in txn between Proks/Euks in terms of space/time?
Txn is compartmentalized in Euks -> the RNA transcript must be transported from the nucleus before translation can begin. Poses a point for regulation.
What are exons?
Coding regions in eukaryotic monocistronic RNA.
What is the eukaryotic promoter region?
the TATA Box
What are introns?
Intervening regions that get excised from monocistronic RNA.
What is the initiation event of eukaryotic txn?
-Incorporation of the CAP structure.
Where is the TATA box located?
-30 upstream
What is the CAP structure?
7-methyl guanosine
What prokaryotic sequence is the TATA box analogous to?
The pribnow box (-10)
What does 7-methyl guanosine consist of?
-Methylated guanosine
-5'-5'triphosphate
-2 riboses
What are the functions of the 5' CAP on mRNA?
1. Enhanced stability of mRNA protected from nucleases
2. Enhanced translation efficiency
What eukaryotic sequence is analogous to the prok -35 promoter sequence?
The -80 CCAT sequence.
What enzyme is the CAP created by?
Guanylyltransferase
So what is the eukaryotic promoter region made up of?
Two consensus regions:
-80 CCAT
-30 TATA
What are exons?
Coding regions in eukaryotic monocistronic RNA.
What are introns?
Intervening regions that get excised from monocistronic RNA.
What is the initiation event of eukaryotic txn?
-Incorporation of the CAP structure.
What is the CAP structure?
7-methyl guanosine
What does 7-methyl guanosine consist of?
-Methylated guanosine
-5'-5'triphosphate
-2 riboses
What are the functions of the 5' CAP on mRNA?
1. Enhanced stability of mRNA protected from nucleases
2. Enhanced translation efficiency
What enzyme is the CAP created by?
Guanylyltransferase
How are the riboses (sometimes) methylated in the 7-methyl guanosine CAP?
By Methyl transferases, using SAM as a donor.
What types of RNA are capped?
Only one! mRNA.