10/29

Question 1

Why is the lack of 3’-5’ exonuclease activity in RNAPs not an issue?

Answer 1

This is not a problem bc mRNAs have a short half life, which means that mutant mRNAs do not survive that long. This characteristic is important in the evolution of viruses w RNA genomes.

Question 2

Termination (transcription):

Answer 2

Termination (transcription): transcription proceeds until the RNAP runs across a terminator sequence

• terminator facilitates dissociation of the RNAP from the DNA template and subsequent release of the synthesized mRNA
• terminator is located downstream of gene coding sequence

Question 3

In bacteria, there are two types of transcriptional termination:
1)
2)

Answer 3

1) self termination (Rho independent)

2) Rho dependent

Question 4

-
-

Answer 4

Self termination: dependent only on the characteristics of the sequence of the DNA template at the termination site
(-Characteristics include:
G/C rich region followed by a series of or more Adenines in the template DNA
G/C rich region is palindromic (same sequence forwards and backwards) and arranged as inverted repeats)
-when inverted repeats are transcribed into a ssRNA, the complementary bases fold back onto each other to form a hairpin
-destabilizing hairpin tugs on the RNAP as it’s trying to transcribe DNA template
-hairpin causes mRNA to fall off bc of weak pairing. Release of transcript and termination of transcript.

Question 5

Rho dependent termination:
1)
2)
*differences btwn self termination

Answer 5

Rho dependent termination: catalytic event that involves Rho protein enzyme

1) Rho is a Helicase that enzymatically breaks H-bonds btwn mRNA transcript and its DNA template
2) terminator is G/C rich but not necessarily palindromic and not followed by a series of Adenines

Question 6

Rho dependent termination (process):
1)
2)
3)
4)
5)

Answer 6

1) Rho protein binds to a rut site (Rho utilization) on the 3’ end of the mRNA
2) Rho protein then moves down mRNA transcript following behind the RNAP
3) RNAP slows down when it reaches GC rich area bc it is harder to transcribe through the strong bonds
4) this allows Rho protein to catch up to RNAP
5) at that point it enzymatically breaks H-bonds holding mRNA to template. This causes the release of mRNA and termination of transcript.

Question 7

Eukaryotic transcription:

Answer 7

Eukaryotic transcription: more proteins involved called transcription factors

Question 8

Transcription factors (TF):

Answer 8

Transcription factor: any protein that interacts w transcriptional machinery and RNAP to increase or decrease the rate of eukaryotic transcription

Question 9

________ transcribed mRNA.

Answer 9

RNAPII transcribes mRNA.

Question 10

_____________ takes place in the nucleus, _____________ happens in cytoplasm.

Answer 10

Transcription takes place in the nucleus, translation takes place in the cytoplasm.

Question 11

Transcription and translation are physically separated by the nuclear membrane: aka they are ___________.

Answer 11

Uncoupled transcription and translation.

Question 12

Eukaryotic transcripts:

Answer 12

Eukaryotic transcripts: monocistronic

Question 13

Prokaryotic transcripts:

Answer 13

Prokaryotic transcripts: may be polycistronic

Question 14

Exons:

Answer 14

Exons: transcribed and translated

Question 15

Introns:

Answer 15

Introns: only transcribed

Question 16

The promoter region (RNAP binding site) is more involved than in prokaryotic transcription and requires more accessory proteins called:

Answer 16

The promoter region (RNAP binding site) is more involved than in prokaryotic transcription and requires more accessory proteins called: transcription factors

Question 17

What is a promoter in eukaryotic transcription made of?

Answer 17

Promoter is made of:
- -90 site (GC box)
- -75 site (CAT box) 
- -25 site (TATA box)
TATA box is analogous to Pribnow box: is AT rich and facilitates unwinding

Question 18

Regulatory elements

Answer 18

Regulatory elements are the site in the DNA that can be bound by TFs that can then influence the rate of transcription

Question 19

Enhancers

Answer 19

Enhancers: regulatory elements (binding site for transcription factors) that when bound by activators (type of TF) increase the level of transcription

Question 20

Activators are _________ TFs

Answer 20

Activators are positive TFs

Question 21

Enhancers act to……

Answer 21

Enhancers act to stimulate RNAP binding to the promoter by stabilizing the initiation complex

Question 22

The location of enhancers are relatively _____________________.

Answer 22

The location of enhancers are relatively position independent

Question 23

Silencers:

Answer 23

Silencers: regulatory elements similar in properties to enhancers. Eg: position independent.
-except silencers are bound by TFs called depressors that serve to decrease/inhibit transcription by destabilizing or interfering w RNAP binding to the promoter

Question 24

RNAPs do not have ____________ activity.

Answer 24

RNAPs do not have proofreading activity. This means they do not have 3’-5’ exonuclease activity.

Question 25

Eukaryotic mRNA processing

Answer 25

Pre mRNA ---processing---> mature mRNA

Question 26

In eukaryotes, transcription is separated from translation by the _______________. (_____________. )

Answer 26

In Eukaryotes, transcription is separated from translation by the nuclear membrane. (Uncoupled.)

Question 27

Precursor mRNA that is processed to mature mRNA ---> transported to ____________ ---> translated

Answer 27

Precursor mRNA that is processed to mature mRNA ---> transported to cytoplasm ---> translated

Question 28

Three primary processing steps: 1) 2) 3)

Answer 28

1) 5' capping 2) 3' polyadenylation 3) splicing

Question 29

5' capping:

Answer 29

5' capping: shortly after transcription begins, there is an addition of 7-methylguanasine cap to 5' end of mRNA. Serves two functions: 1) protects 5' end from degradation 2) MeG cap serves as the ribosome binding site for translation NO CAP=NO TRANSLATION

Question 30

3' polyadenylation:

Answer 30

3' polyadenylation: the addition of 50-300 Adenines to 3' end of mRNA. Serves two functions: 1) protects 3' end from degradation 2) involved in termination of transcript - enzyme responsible for 3' polyadenylation is called Poly A polymerase (PAP)

Question 31

Enzyme responsible for 3' polyadenylation

Answer 31

Enzyme responsible for 3' polyadenylation is called PolyA polymerase (PAP). Can add on bases w/o needing a template.

Question 32

Splicing:

Answer 32

Splicing: removal of non coding introns and joining of the coding exons

Question 33

If exons are improperly joined...

Answer 33

If exons are improperly joined mutant / abnormal mRNA Is formed that will make a mutant or nonfunctional protein

Question 34

Conserved sequences found at the junction sites of introns/exons boundaries are recognized by splicing proteins called _________.

Answer 34

Conserved sequences found at the junction sites of introns/exons boundaries are recognized by splicing proteins called snRNPs. (Small nuclear ribonucleoproteins)

Question 35

Spliceosome

Answer 35

Spliceosome: together the snRNPs associate w pre mRNA to form a splicing complex called a spliceosome.

Question 36

``` Steps to splicing 1) 2) 3) 4) 5) (two steps) ```

Answer 36

1) U1 attaches to the 5' splice site of exon 1 2) U2 attaches to a beach point adenine located w/in intron 3) U4, U5, U6 associate to bring exons into close proximity 4) realize of U1 and U4 results in formation of an active spliceosome 5) two step transesterification reaction where you: 1) have cleavage at 5' splice site of exon 1 by nulceophilic attack of 2'OH group of branch point adenine on 5' PO4 group of exon 1. 2) the now exposed 3'OH group of exon 1 carries out nulceophilic attack of phosphate group of exon 2

Question 37

Ribozymes

Answer 37

Ribozymes: self splicing RNAs... RNA enzyme

Question 38

Why have introns? 1) 2) 3)

Answer 38

1) introns protest genes from errors in meiotic recombination (higher chance of errors occurring in large introns) 2) exon shuffling: rearrangement of exons at DNA level (aka inherited). Leads to new genes. Important to evolution and adaptation. 3) differential splicing: rearmament of exons at RNA level (aka not inherited). Important bc it greatly increases coding capacity of genome.