W4 L1 Regulation of Transcription Initiation: The GAL system M Flashcards
Galactose utilization in gene S. Cer
-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
Other galactose regulation
-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
Chromatin Immunoprecipitation (ChIP) chip
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
Chipsequase
-identify the protein bounded to DNA of interest
-sequences the gene, find out the area of binding
Identifying regulatory gene
- if a gene mutation affect man other gene, it might be a regulatory gene
mutation of a positive regulator
-common case: recessive, non-inductable
-rare case: dominant, constitutive
Mutation of negative regulator
-common: recessive, constitutive
-rare: dominant, non-inductable
How to work out the order of regulatory gene
-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
Gal4 gene in dept
-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
gene role in the galactose pathway
! Structural genes
! GAL1, GAL2, GAL7, GAL10 and MEL1
! Regulatory genes
! GAL4, GAL3, GAL11 and GAL80
How to figure out the location of domain in the gene
-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
Gal 4 regconiction
-bind as a dimer to CCG>N11>CCG at the 5’ and 3’ end
Gal 3 and Gal 11 mutant analysis
-gal3-: recessive, slower induction, common
-gal11-: recessive, reduced induction, common
So what is Gal3
-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
Role of Gal 80
Gal80 binds to Gal4 which Blocks activation
! Gal3 bound to galactose relieves Gal80 mediated inhibition
Two model of GAL3 to GAL80 regulation
A: Gal 3 temporary enter the nucleus and bind to GAL80
B:GAL3 recruit gal 80 out to the cytoplasm (most likely)
GAL11
-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
The whole picture of Gal pathway
- Gal4 is bound to UASGAL under non-
inducing (no galactose) conditions, but kept inactive by interaction with Gal80; - Upon galactose induction, Gal80 leaves
the nucleus, allowing Gal4 to recruit SAGA; SAGA (Gcn5) – HAT complex - Mediator binds, followed by;
- The recruitment of Pol II and the general transcription machinery, initiating transcription;
- 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.
Regulatory protein and chromatin modification
DBD proteins can recruit chromatin modifying complexes to control transcription initiation
-make a gene repressive via histone deacetylase
-histone methyl transfer affect gene transcription
The whole initiation pathway
