transcription termination and tailing

Question 1

to terminate a sequence, what must RNA polymerase do?

Answer 1

stop incorporation at the proper place, release the RNA and disengage from the DNA strands

Question 2

are terminators 100% effective?

Answer 2

nope!

Question 3

what are the 2 types of termination?

Answer 3

intrinsic and rho dependent

Question 4

what is intrinsic termination?

Answer 4

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

Question 5

what is rho dependent termination?

Answer 5

requires a different termination process and the action of the Rho protein

Question 6

what is Rho specific to?

Answer 6

sequences that mediate its recruitment, sequences that antagonize its activity, and determinants of RNA polymerase pairing

Question 7

do rho dependent terminators have the same characteristics as intrinsic terminators?

Answer 7

no. they have no string of Us or hairpins

Question 8

what is Rho?

Answer 8

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

Question 9

what are the 2 main features of bacterial intrinsic termination?

Answer 9

an inverted repeat sequence forming a hair pin and a string of 8-10 uracils

Question 10

what are some characteristics of bacterial intrinsic termination?

Answer 10

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

Question 11

what is an attenuator?

Answer 11

a transcription terminator that occurs at the start or in the middle of an operon rather than at the end

Question 12

how does termination by RNAP2 occur?

Answer 12

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

Question 13

what are the 2 models of termination by RNAP2?

Answer 13

allosteric and torpedo

Question 14

describe the allosteric model;

Answer 14

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

Question 15

describe the torpedo model;

Answer 15

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

Question 16

is it possible to have a mix of allosteric and torpedo model?

Answer 16

yes

Question 17

describe termination by RNAP1;

Answer 17

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

Question 18

describe termination by RNAP3;

Answer 18

Intrinsic terminator sites are recognized by RNA pol 3. These have a stretch of Us

Question 19

what are the ways a polynucleotide can be digested?

Answer 19

cleaved internally and digested progressively from one of the ends (exo and endonucleases)

Question 20

what is an exonuclease?

Answer 20

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

Question 21

what is an endonuclease?

Answer 21

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.

Question 22

what did Mary Edmunds discover?

Answer 22

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.

Question 23

describe the characteristics of Polyadenylation;

Answer 23

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

Question 24

describe the formation of the poly(A) tail;

Answer 24

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)

Question 25

what does capping do?

Answer 25

needed to permit RNAP2 to continue transcription elongation

Question 26

what is polyadenylation for?

Answer 26

needed to prepare the mRNA transcript for transport to the cytoplasm

Question 27

what is the combination of the 5’ cap and 3’ tail used for?

Answer 27

for efficient ribosome binding and initiation of translation

Question 28

why are some bacterial RNAs resistant to digestion?

Answer 28

because they have 3’ stem loops. The addition of a poly-A tail allows an exonuclease to destabilize the stem loop and digits it bit by bit.