transcription termination and tailing Flashcards
to terminate a sequence, what must RNA polymerase do?
stop incorporation at the proper place, release the RNA and disengage from the DNA strands
are terminators 100% effective?
nope!
what are the 2 types of termination?
intrinsic and rho dependent
what is intrinsic termination?
a mechanism that depends only on the presence of repeat sequences in the RNA transcript to form a secondary structure that is needed for and triggers termination
what is rho dependent termination?
requires a different termination process and the action of the Rho protein
what is Rho specific to?
sequences that mediate its recruitment, sequences that antagonize its activity, and determinants of RNA polymerase pairing
do rho dependent terminators have the same characteristics as intrinsic terminators?
no. they have no string of Us or hairpins
what is Rho?
a hexameric ATPase that can form an open or closed structure. It binds ro C rich areas called rho utilization sites. Has an open structure before binding and once bound it closes and ATP hydrolysis pulls the RNA through the ring and away from RNA polymerase
what are the 2 main features of bacterial intrinsic termination?
an inverted repeat sequence forming a hair pin and a string of 8-10 uracils
what are some characteristics of bacterial intrinsic termination?
Functions in the absence of any other factors
Function in the RNA molecule that is being transcribed
The stem-loop/hair pin is GC rich and causes destabilization
what is an attenuator?
a transcription terminator that occurs at the start or in the middle of an operon rather than at the end
how does termination by RNAP2 occur?
New mRNA is cleaved at the 3’ end
Processed mRNA is polyadenylated before being sent to cytoplasm
Termination of RNAP 2 genres is couples to processing of the 3’ end of the mRNA
The mRNA is cleaved at the poly-A signal (AAUAAA) and the 3’ end of the mRNA is processed by adding the poly-A tail
RNAP2 continues to transcribe after the polyadenylation signal
what are the 2 models of termination by RNAP2?
allosteric and torpedo
describe the allosteric model;
RNA processing proteins associated with cleavage and/or polyadenylation cause a conformational change in the RNA polymerase
This change results in dissociation of RNAP2 from DNA
describe the torpedo model;
After cleavage of the mRNA, the leftover RNA downstream of the poly-A site is digested by a 5’ to 3’ exonuclease (Rat1)
The exonuclease continues to degrade the new RNA until it bumps into the RNAP
The exonuclease acts as the torpedo that disrupts RNA synthesis and causes the enzyme to dissociate from the DNA
is it possible to have a mix of allosteric and torpedo model?
yes
describe termination by RNAP1;
Termination for RNA polymerase 1 requires additional proteins. Poly-U sequences are present, but a DNA binding protein (Reb1p in yeast and TIIF1 in mice) binds to sequences downstream of the transcription unit
describe termination by RNAP3;
Intrinsic terminator sites are recognized by RNA pol 3. These have a stretch of Us
what are the ways a polynucleotide can be digested?
cleaved internally and digested progressively from one of the ends (exo and endonucleases)
what is an exonuclease?
digest a polynucleotide from the ends (usually 3’) the Rat1 enzyme is a 5’ exonuclease. This can generate molecules with long overhanging single stranded ends
what is an endonuclease?
cleave a polynucleotide internally. May recognize a specific sequence or element. Can be specific for ssRNA or dsRNA. RNase 3 and RNase P are endonucleases. May result in different outcomes. If it cleaves it at the same place on both strands it will make a blunt end. If they cleave asymmetrically on each strand then an overhang is created.
what did Mary Edmunds discover?
key discoveries regarding the processing of mRNA. Used HeLa cells, there was an uncertainty about the relationship between hnRNA and mRNA and had the idea that mRNA had a poly-A tail.
describe the characteristics of Polyadenylation;
The 3’ end have approx. 200-250 adenosines added (poly-A tail)
It protects eukaryotic mRNA from nuclease degradation
Plays an essential role in the initiation process of protein synthesis
In bacteria: much smaller poly-A tail added and it enhances degradation
Transcription past the 3’ end-processing signals leads to recruitment of the 3’ end-processing complex
describe the formation of the poly(A) tail;
First: the AAUAAA signal is recognized by cleavage and polyadenylation specificity factor (CPSF - 3 subunits). The cleavage stimulation factor (CstF) and an endonuclease are recruited and the RNA is cleaved.
Finally: poly-A polymerase catalyzes the addition of approx. 200 adenosines. Once added the poly-A tail is bound by poly-A binding protein (PABP)
