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58 Cards in this Set

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What is a transcription unit?
A transcription unit is the area of DNA where the gene, or genes, that will be transcribed are located.
In relation to the +1 base, where is the TATA box located? What is the TATA box?
The TATA box is a promoter and it is located upstream of the transcription unit.
What is DNA melting?
When the two strands of dsDNA come apart into individual strands.
Which direction does RNAP travel on the template DNA?
Downstream
What is the small region of dsDNA that is melted called during transcription?
The transcription eye
What are the three functions of the promoter?
1. Determination of the +1 base.
2. Choice of the template strand and overall direction of transcription.
3. Control of the frequency of transcription (strong vs. weak promoters)
What are the three steps in transcription?
Initiation, Elongation, and Termination
True or false: RNAP is DNA-dependent.
True
What order should the following basic initiation steps be in?
a. Open complex formation (local melting of DNA at the +1 base)
b. Initial RNA synthesis
c. Promoter binding by RNAP to form closed complex.
C, A, B
In bacterial RNAP, what are the five core subunits that make up the RNAP holoenzyme?
alpha (2), beta, beta prime, and omega (w)
In the RNAP holoenzyme, what subunit can be exchanged and is not one of the five core subunits?
Sigma
Where is the active site on bacterial RNAP?
On the beta prime subunit
Which subunit on RNAP recognizes different sets of promoters and allows RNAP to find the proper sequence?
Sigma
What RNAP subunit dissociates during elongation?
Sigma
Why is it beneficial that in bacterial RNAP, the sigma subunit can be exchanged for a different sigma subunit?
Because this allows RNAP to recognize and transcribe a new set of sequences without having to build an entirely new RNAP.
True or False: RNAP and DNAP both have proofreading 3'-5' exonuclease.
False, RNAP does not.
How many types of RNAPs exist in the prokaryotic cell?
One
In the prokaryotic cell, what does RNAP require for transcription to occur?
DNA template, dNTPs (ATP, GTP, UTP, CTP) and Mg2+
What are dNTPs?
Deoxy nucleotide triphosphates; required for bacterial transcription
In what direction does transcription occur, 5' to 3' or 3' to 5'?
5' to 3'
In bacteria, what does the 5' end of mRNA carry?
A triphosphate (because of ATP).
How many phosphate groups are used to link two sugars in the sugar-phosphate backbone of DNA and RNA?
One phosphate group
What two cofactors are need during the construction of the sugar-phosphate backbone in DNA?
Asp(AA) and Mg2+(ion)
During transcription, when one base is attached to another, how many phosphate groups are lost?
Two - one diphosphate is lost (from ATP - one remains)
During construction of the new RNA strand, what carbon does the phosphate group bond to?
The 3' carbon
How can the binding of DNA to proteins be measured (can show RNAP binding to the promoter sequence)?
DNA footprinting
What are the four steps in DNA footprinting?
1. A solution of identical DNA fragments are radioactively labeled at one end.
2. The solution is treated with DNase until each strand is cut once (on average) - no cuts are made where RNAP is bound.
3. The DNA fragments are isolated and denatured. Only labled DNA are detected in the next step.
4. The fragments are separated by polyacrylamide gel electrophoresis and the radiolabeled bands are visualized on x-ray film. The missing bands indicate where RNAP was bound to DNA.
How many base pairs long is the transcriptional eye? How many base pairs are part of the RNA/DNA heteroduplex?
The transcriptional eye is 17 bps long, and the RNA/DNA heteroduplex is 8 bps long.
Discovered by DNA footprinting.
What site is located on bacterial RNAP's beta prime subunit?
The active site, where the next base will be added during transcription.
What is another name for the nontemplate strand of DNA?
The coding strand
In what direction is transcription occurring (on the template)?
5' to 3', downstream
What are activator proteins?
Proteins that facilitate RNAP binding to a specific promoter - increases the frequency of transcription initiation.
What are repressor proteins?
Proteins that block RNAP sythesis from specific promoters - reduce the frequency of transcription initiation
True or false: A ribosome is made of protein.
False - it is made of RNA
What does RNAP I produce in eukaryotes?
pre-rRNAs (28S, 18S, and 5.8S)(ribosomes)
What does RNAP II produce in eukaryotes?
mRNAs (primarily)
What does RNAP III produce in eukaryotes?
tRNAs, 5S rRNA
True or False: RNA can catalyze reactions.
True
Reading the template DNA strand from 5' to 3', which comes first, the promotor/TATA box or the +1 base?
The promoter/TATA box (upstream from +1 base)
How many subunits are in the core of Pol II?
Twelve; the main three are:
1. RBP1 (largest, shows homology to E.coli's beta prime)
2. RBP2 (homologous to E.coli's beta)
3. RPB3 and 11 (homologous to E.coli's alpha)
What is a possible reason to explain why eukaryotic Pol II is more complex than RNAP?
Because eukaryotic genomes are larger and more tightly packaged than prokaryotic genomes.
True or False: Pol II can bind to a gene by itself.
False; needs the prior binding of proteins called transcription factors
In what order do the transcription factors bind in eukaryotic transcription?
1. TBP (recognizes TATA box/promoter site) and TFIIB (sometimes TFIIA)
2. TFIIF brings Pol II to the site.
3. TFIIE
4. TFIIH
The Big Frog Eats Hornets
Approximately where is the TATA box located on the template DNA?
About 25 to 30 base pairs upstream of the +1 base.
Approximately where is the enhancer located on the template DNA?
Usually hundreds or thousands of base pairs upstream of the promoter.
It's farther than you think.
What are the steps in eukaryotic transcription?
1. TFIID arrives at the transcription bound to TBP, which locates and binds to the promoter region/TATA box.
*The binding of TBP creates a bend in the DNA molecule.
*Sometimes TFIIA binds to aid in the binding of TFIID
2. TFIIB interacts with the TFIID complex and soon, the Pol II complex.
3. Pol II and TFIIF form a complex, then bind to the DNA.
4. TFIIE binds to the Pol II complex to create a docking site for TFIIH.
5. TFIIH binds to the docking site. A closed complex is formed.
6. To activate the unwinding of DNA, ATP gives one phosphate per molecule of DNA (leave ADP).
7. Elongation begins and the general transcription factors are released (TFIIE, TFIIA).
8. TFIID/TBP are still bound to the TATA box when elongation ends.
9. The DNA 'eye' closes and the mRNA and phosphates are released from the Pol II complex.
10. The Pol II complex assumes its original conformation and leaves the DNA.
11. The TFIID complex releases by way of TBP, and the DNA returns to its original conformation.
True or False: TFIID has more than one subunit.
True; TFIID has eleven TBP-associated factors and a single TBF (TATA box-binding factor).
What are the activities of TFIIH?
1. DNA Helicase - enzyme for the formation of open and closed complexes (DNA unwinding)
2. Protein kinase - triggers elongation (phosphorylates)
When does termination occur in prokaryotes?
Termination occurs when the polymerase reaches a terminator sequence.
True or False: Termination of transcription can be determined entirely by looking at the DNA sequence during transcription.
False; many terminators require a hairpin to form in the secondary structure of the RNA being transcribed.
What is antitermination?
When the enzyme continues transcription past the terminator sequence (an event called readthrough)
What are the two types of bacterial transcription termination?
1. Rho-dependent (works on DNA and RNA); the rho RNA:DNA helicase (ancillary factor)disrupts the transcription bubble
2. Rho-independent;
a. structure of the RNA:DNA heteroduplex is less stable at certain sequences, which predisposes the bubble to disassemble.
b. self-complimentary sequence in the transcript followed by a run of U's (just prior to 3' end of the transcript)
Rho-independent termination
No ancillary factor is required.
Structure of the RNA:DNA heteroduplex is less stable at some sequences, causing the polymerase to pause. The pause typically lasts -60 seconds and predisposes the transcription bubble to disassemble.
Self-complimentary sequence in the transcript followed by a run of U's, just prior to the 3' end.
What three things do bacterial initiation and termination have in common?
1. Both are subject to control/regulation.
2. Both require breaking of hydrogen bonds (initiation: melting of dsDNA; termination: RNA-DNA dissociation)
3. Both require additional proteins to interact with the core enzyme.
What is one main difference between initiation and termination (besides the obvious ones)?
Termination involves recognition of signals in the RNA transcript or by ancillary factors.
What are the only molecules in the cell that can read the genetic code?
tRNA
Before interacting with mRNA, tRNA must be ________. How does this occur?
Activated; Occurs when one of the 20 different aminoacyltRNA sythetases links its corresponding amino acid to the 3' end of the tRNA molecule.
tRNA contains a series of _________ _________ that allow the _________ of the linear RNA molecule into a cloverleaf configuration.
Inverted sequences; folding