W4 L1 Regulation of Transcription Initiation: The GAL system M

Question 1

Galactose utilization in gene S. Cer

Answer 1

-screening from a gal-mutant
-found out the existence of Gal1, Gal2, Gal5,Gal7, Gal10 and MEL1
-MEL1 turn alpha-galactosidase into galactose
-Gal2 is a permease, allow galactose to enter
-Gal1, 7,5,10 turnit into TCA

Question 2

Other galactose regulation

Answer 2

-Glucose are able to bind to GAL 1,2,3,7,10
-while galactose can bind to all the transcription factor
-showed that many gene are involve that are invisible to complimentary analysis

Question 3

Chromatin Immunoprecipitation (ChIP) chip

Answer 3

Determining DNA sequence bound by proteins
-cross link protein to DNA in living cell with formaldehyde, protein that bind to DNA remain in place
-break open cell and smear dna
-add primary antibody of interest
-add antibody binding beads
-immunoprecepitate to enrich for fragments bound by protein of interest
-reverse cross link and treat with proteinsae K
-detect and quantify the DNA for analysis

Question 4

Chipsequase

Answer 4

-identify the protein bounded to DNA of interest
-sequences the gene, find out the area of binding

Question 5

Identifying regulatory gene

Answer 5

• if a gene mutation affect man other gene, it might be a regulatory gene

Question 6

mutation of a positive regulator

Answer 6

-common case: recessive, non-inductable
-rare case: dominant, constitutive

Question 7

Mutation of negative regulator

Answer 7

-common: recessive, constitutive
-rare: dominant, non-inductable

Question 8

How to work out the order of regulatory gene

Answer 8

-create a double mutant of both recessive or both dominant mutation of the regulatory factor
-look at the final result, which phenotype the double mutant is expressing is the one that is downstream

Question 9

Gal4 gene in dept

Answer 9

-positive acting regulator with a DNA binding domain and 2 activation domain
-can bind to GAL 1,7, 10, promoter which have a GAL4 binding domain

Question 10

gene role in the galactose pathway

Answer 10

! Structural genes
! GAL1, GAL2, GAL7, GAL10 and MEL1
! Regulatory genes
! GAL4, GAL3, GAL11 and GAL80

Question 11

How to figure out the location of domain in the gene

Answer 11

-Using mutation, chopping pieces of a little at a time
-work out when a piece is gone, does they still bind and can they still regulate correctly

Question 12

Gal 4 regconiction

Answer 12

-bind as a dimer to CCG>N11>CCG at the 5’ and 3’ end

Question 13

Gal 3 and Gal 11 mutant analysis

Answer 13

-gal3-: recessive, slower induction, common
-gal11-: recessive, reduced induction, common

Question 14

So what is Gal3

Answer 14

-signaling molecule, use to regconise galactose
-slow induction due to lack of binding
! Signaling molecule that binds galactose
! Binds Gal80 when bound to galactose
! Cytoplasmic localisation after binding
Believe to originated from a paralegal of GAL1 which does share some small fuction son not a total loss of fuction

Question 15

Role of Gal 80

Answer 15

Gal80 binds to Gal4 which Blocks activation
! Gal3 bound to galactose relieves Gal80 mediated inhibition

Question 16

Two model of GAL3 to GAL80 regulation

Answer 16

A: Gal 3 temporary enter the nucleus and bind to GAL80
B:GAL3 recruit gal 80 out to the cytoplasm (most likely)

Question 17

GAL11

Answer 17

-gal11- - recessive, reduced induction, common
-the MED15 mutant showed that it interact GAL4 to recruit TF2D and RNApol2, acting as a part of the mediator complex

Question 18

The whole picture of Gal pathway

Answer 18

1. Gal4 is bound to UASGAL under non-
   inducing (no galactose) conditions, but kept inactive by interaction with Gal80;
2. Upon galactose induction, Gal80 leaves
   the nucleus, allowing Gal4 to recruit SAGA; SAGA (Gcn5) – HAT complex
3. Mediator binds, followed by;
4. The recruitment of Pol II and the general transcription machinery, initiating transcription;
5. Swi/Snf is not absolutely required for activation, but it is found at the promoter in a Gal4-, Mediator- and general transcription machinery-dependent manner.

Question 19

Regulatory protein and chromatin modification

Answer 19

DBD proteins can recruit chromatin modifying complexes to control transcription initiation
-make a gene repressive via histone deacetylase
-histone methyl transfer affect gene transcription

Question 20

The whole initiation pathway

Answer 20