Exam 4 (Ch 29-25) Flashcards
What is the Klenow fragment?
Region of DNA polymerase that contains a DNA binding cleft.
-Prokaryotic and eukaryotic RNA polymerase have similar fragments
What are the 5 functions of RNA polymerase?
- search for cis-acting elements
- Unwind a short segment of double helix
- Selects the correct ribonucleoside triphosphate (NTPs) and catalyzes phosphodiester bond formation
- Detects termination signals that say where transcription ends
- Interacts with activator/repressor proteins (i.e. transcription factors/trans-acting elements) that modulate rate of transcription initiation
What are cis-acting elements?
They are initiation/promoter sites that are on the same molecule as RNA polymerase
What are promoters?
Sequences of DNA that direct RNA polymerase to the proper initiation site for transcription/act as binding sites for transcription factors/accelerate transcription.
What/where are the 2 most common promoter sequences found on prokaryotic DNA?
Where: 5’ side of the start site
What: -10 and -35 sequence (centered 10 and 35 nucleotides upstream of start site)
What are the 3 stages of RNA synthesis?
- Initiation
- Elongation
- Termination
Describe footprinting and its function.
Function: to identify protein binding sites on DNA
How:
1. 32P labeled DNA is (un)treated with RNA polymerase and digested by DNase –> chain gets cut
2. Fragments separated via gel electrophoresis
3. Fragments made in absence of polymerase but not made in presence of polymerase show which bases were blocked by bound protein from hydrolysis
How does the sequence of the DNA coding strand compare to that of the RNA transcript?
They are the SAME with the exception of thymine (T) in place of uracil (U)
How does the sequence of the DNA template strand compare to that of the RNA transcript?
It is complementary to that of the RNA transcript
What are the other names that the coding strand and template strand known as?
Coding Strand= Sense (+) strand
Template Strand= Antisense (-) strand
Does RNA synthesis require a primer to start?
No, it can start de novo.
What is and where is the highly distinctive tag carried by newly synthesized RNA chains?
Where: 5’ end
What: First base at this end is either pppG or pppA
Significance: confirms that RNA synthesis starts at the 5’ end
What are the functions of each subunits of RNA synthetase holoenzyme?
Subunits (5): α2ββ’σ
σ=finds sites on DNA where RNA transcription can start
-breaks off from subunit once ~10 bases have been
assembled and can initiate further transcriptions
-dif types serve dif functions
(core enzyme) α2ββ’=used to complete transcription
Describe the elongation stage of RNA synthesis.
- NTP binds to active site of RNA polymerase
- NTP forms a Watson-Crick base pair with template strand
- Activates 3’-OH group of growing RNA chain
- Which then attacks the α-phosphoryl group
- Creates a phosphodiester bond and displaces PPi
What are the ways that RNA transcription can be terminated?
- Self Termination
- Termination Signals
a. Hairpin structures
b. Rho Factor
How can hairpin structures terminate RNA transcription?
Hairpin structure occurs where there are multiple uridines (U) in a row which is the weakest base pair interaction
-the RNA strand separates from the DNA strand and then from the polymerase thus ending synthesis
How does Rho factor terminate RNA transcription?
Rho factor=an ATPase enzyme
-it slowly pulls the transcript RNA (tRNA) away from RNA polymerase
What are transcription factors?
Proteins required to initiate transcription
What are enhancers/silencers?
Auxiliary nucleotide sequences in the template DNA (i.e. a cis acting element) to which activators and repressors bind
What is the promoter for RNA Polymerase II?
TATA Box
What are the functions of the following transcription factors: –TBP–TAFs–TFIIH–
TBP: a TATA binding protein that causes DNA to unwind and bend
TAFs: modulates rate of transcription
TFIIH: helicase that separates strands
What is a preinitiation complex?
Preliminary structure of RNA polymerase consisting of a conglomerate of transcription factors that assemble around TBP
Describe what occurs in RNA transcription initiation.
- Assembly of preinitiation complex (PIC)
- Phosphorylation of the carboxy terminal domain (CTD) to create an open PIC complex
- Once phosphorylated, CTD recruits RNA-processing enzymes
- RNA polymerase moves away from TATA initiation sequence and maj of transcription factors
- Left behind transcription factors can now assemble another PIC
What are the 3 modifications that occur during posttranslational processing?
- Removal of polynucleotide segments by exo/endo-nucleases
- Addition of polynucleotide segments to the ends
- Modification of specific nucleotide residues
