Lecture 6 - Overview of eukaryotic gene control

Question 1

RNAP I where and what it does

Answer 1

In the nucleolus. Transcribes precursor ribosomal RNA (rRNA)

Question 2

RNAP II what it does (3)

Answer 2

Transcribes messanger RNAs (mRNAs) and four small nuclear RNAs (snRNAs) that take part in splicing + transcribes miRNAs

Question 3

RNAP III what it does (2)

Answer 3

Transcribes transfer RNA (tRNA), 5S RNA, other small stable RNAs including one involved in RNA splicing

Question 4

During chromatography used for protein seperation, the tube contains a _________, and a ________ is continuously applied from the top. Proteins go down at different speed depending on _____________. Each ____ collected is a _________

Answer 4

solid matrix. solvent, their affinity with the matrix (beads), ml. fraction.

Question 5

Three chromatography techniques and what they’re based on

Answer 5

1) Gel filtration chromatography based on size and shape.
2) Ion exchange chromatography based on net charge
3) Antibody-affinity chromatography : binds a specific protein on basis of antibody interaction

Question 6

What happens in gel filtration chromatography

Answer 6

small proteins ‘‘hang up’’ as they’re caught by the polymer beads. Big proteins come out first and small ones come out second

Question 7

What happens in ion-exchange chromatography

Answer 7

+ and - charged proteins. If positively charged bead, neg. charged prots hang up. Positively charged prots (cations) come out first. Rest is eluted with salt solution NaCl. Cl - takes the place of the prots and hangs on the beads. negatively charged prots come out second.

Question 8

What happens in antibody-affinity chromatography

Answer 8

1) Prots specific to antibody hang up (bead with antibody) and non specific ones come out first 2) Elute with pH 3 buffer so that prots w/ antibody affinity come out too

Question 9

Goal of chromatography w/ proteins and what fractions cannot be used

Answer 9

Goal is to obtain proteins with activity. first fractions = junk and you can’t use it.

Question 10

Chrom. : For each fraction collected, we compare the amount of _______ obtained to the ___________. We want fractions with the __________

Answer 10

active protein, relative amount of protein. active proteins

Question 11

How can we purify proteins

Answer 11

Sucessive liquid chromatography 1) Ion-exchange 2) Gel filtration 3) Antibody affinity

Question 12

Northern blot analysis is used with _________ and it can be used (for example) to determine if __________ of RNA was supported by particular _________

Answer 12

RNA. transcription. proteins

Question 13

Steps of northern blotting briefly (similar to RNA)

Answer 13

1) Extraction and cleaving with restriction enzymes 2) Gel electrophoresis 3) Blotting (nitrocellulose, alkaline solution, paper filter) 4) Incubate nitrocellulose with labelled probes 5) Visualize hybridized probes on X-ray film

Question 14

Substance that can interfere with RNA synthesis and what it is made of. What substance it interacts with and how

Answer 14

alpha-Amanitin. cyclic peptide of 8 amino acids. High activity with Pol II (interacts in its active site)

Question 15

How can you isolate Pol I, Pol II, PolI III and what are their different sensibilities to alpha-amanitin (1 and 10 microgram/ml)

Answer 15

column chromatography. Pol I resists 1 and 10, Pol II can doesn’t resist 1, Pol III resists 1 but not 10

Question 16

Comparison of bacterial and eukaryotic pol (or pol II)

Answer 16

Bacterial RNAP -> 5 subunits. Yeast RNAP II -> 12 subunits

Question 17

2 particular parts in polymerases and their functions

Answer 17

wall and clamp. DNA goes in between wall and clamp. During elongation, clamp stops complex from returning in initiation phase

Question 18

5 subunits of RNAP in E.coli. which ones are necessary and which one is not (but what it helps in)

Answer 18

Beta, beta prime, alpha I, alpha II, omega. Alpha ones necessary. omega subunit is not essential but helps in holding complex together

Question 19

Eukaryotic polymerases, their common subunits

Answer 19

B’-like and B-like subunits. 2 alpha like subunits. 1 omega like subunit. 4 common subunits

Question 20

How eukaryotic RNAPs differ

Answer 20

RNAP I -> 5 additional enzyme-specific subunits RNAP II -> 3 ‘’ RNAP III -> 7 ‘’

Question 21

largest subunit of Pol II and what does it have

Answer 21

beta prime like subunit has CTD tail (carboxy-terminal domain)

Question 22

CTD tail what is it made of

Answer 22

52 nearly identical repeats of Tyr - Ser - Pro - Thr - Ser - Pro - Ser

Question 23

RNA polymerase molecules that initiate transcription have _________________ CTD.

Answer 23

unphosphorylated

Question 24

RNA polymerase molecules that are actively transcribing have a ________________ CTD.

Answer 24

phosphorylated

Question 25

A phosphorylated CTD tail is associated with _____________

Answer 25

elongation

Question 26

On image with red and green DNA, what is green (and marker for what). what is red what is associated with green. what are the fat parts of the chromosome (puffs) and their color.

Answer 26

red-> phosph. green-> unphosph.+ therefore marker for DNA. puffs = a lot of transcription occurs. they are red (phosph. CTD)

Question 27

what is yellow on green/red image

Answer 27

close regions where one is being transcribed and one is not

Question 28

which fungi does alpha amanitin

Answer 28

Amanita phalloides

Question 29

different sensitivities of RNAPs to different concentrations of alpha amanitin

Answer 29

Pol I insensitive to 1 or 10 microg/ml. Pol II sensitive to 1 microg/ml and Pol III insensitive to microg/ml but sensitive to microg/ml