MIMM 323 Lecture 30.docx

5 Pages
Unlock Document

Microbiology and Immun (Sci)
MIMM 323
Caroline Petit- Turcotte

MIMM 323 Lecture 30 Nov. 16  If you have a haploid strain with mutation in alpha1 (mat alpha1-) - No alpha1 produced but alpha2 still produced o Haploid-specific genes are ON o A-specific genes are OFF o Alpha-specific genes are OFF  No receptor and no pheromones released  STERILE STRAIN  Alpha1, alpha2, a1: protein-DNA complexes - Need another protein for expression of alpha and a-specific genes o MCM1, also known as PRTF  Binds upstream activating sequences of alpha and a-specific genes  Called “P-boxes” o In a-cells:  MCM1 binds with high affinity to P-box upstream of a-specific genes   Expression of a-specific genes o In alpha-cells:  The P-box binding site for MCM1 has low affinity recognition (“faulty P-box”)  In order to bind with high affinity, need secondary alpha1 factor  Alpha1 and MCM1 form complex which binds together to P-box   Expression of alpha-specific genes  IN ADDITION, a-specific genes must be repressed:  Alpha2 forms complex with MCM1 (alpha2-MCM1-alpha2) Figure 1: Combinations of PRTF, alpha1 and alpha2  Prevent binding of MCM1 to P-box of a-specific genes   Repression of a-specific genes o In diploid cells:  Alpha2 and alpha1 form a complex (alpha2-alpha1-alpha2)   Repression of haploid-specific genes  Expression of MAT and repression of HML and HMR on chromosome 3 - Why are HMR and HML not expressed in contrast to MAT? o HML and HMR are flanked by silencer sequences (known as “E” and “I”)  Found in higher eukaryotes  Function at a distance o The MAT locus is not flanked by silencers  Known as the “active cassette”, as there is no silencing of gene expression o o o o o o o o Figure 2: Silencers flanking HML and HMR o A deletion of E and I results in expression of BOTH HML and HMR - Many genes required to repress HML and HMR: o SIR1 to 4  SIR = Silent Information Regulator o RAP1  DNA-binding protein; specifies chromatin structure (loose or tight) o Histone H4 - Necessary proteins identified by screening of mutants: o Create mutant for protein, e.g. sir2-  HML, HMR and MAT ALL EXPRESSED   No expression of alpha-specific genes   No expression of a-specific genes   No expression of haploid genes  Although still haploid, behaves like a diploid  Cannot mate  STERILE STRAIN  Thus  Sir2 is negative regulator of expression of HMR or HML  Repression is linked to chromatin structure - The nucleosome = the basic repeating unit of chromatin o Nucleosome core (DNA wrapped around octamer of histones) composed of:  DNA  Two H2A  Two H2B  Two H3  Two H4 o Complete nucleosome composed of: Figure 3: Nucleosome structure; histone octamer,  Nucleosome core with DNA strands linked by H1 histone  One H1 linker histone - Nucleosomes act as a barrier, blocking binding of transcription factors to DNA o For any binding site;  If use naked (unwrapped) DNA to measure TF binding  affinity will be relatively high  Contrast to same piece of DNA wrapped around nucleosome  affinity will be much lower o Thus  can control access of TF to DNA by making changes in chromatin structure - The N-termini of histones are lysine-rich, extend out of the nucleosome, and are subject to modifications: o Residues can be phosphorylated, ubiquitinated, acetylated, methylated, etc.  Modifying enzymes are known as “writers”  Proteins that interact with modified histones and recruit TFs are known as “readers” - Acetylation of lysine residues results in change from positively charged to neutral o Neutralization of the charge  looser nucleosome structure o Easier for TF to access DNA Figure 4: Mechanism of lysine acetylation  Hyper-acetylated histones associated with gene expression  Hypo-acetylated histones associated with gene repression - Mechanism of silencing: o Rap1 and Sir proteins form complex at HML, HMR, telomeres  Rap1 binds to silencer DNA sequences  Allows formation of Sir2, 3 and 4 complex  Sir2 = histone deacetylase - Enzymatic activity is found in Sir2 Figure 5: Silencing mechanism with Rap1 and Sir complex at telomere - Deacetylation of histone residues results in change in chromatin structure from open  closed - Closed chromatin around HML and HMR sequences  genes not expressed  Sir3 and 4 interact with histones o At MAT locus:  No silencer  No formation of Rap/Sir complex  expression of MAT locus gene - To prove that acetylation of lysine residues is responsible for gene expression, perform experiments: o Mutate lysine to arginine  Arginine is positively charged amino acid; can’t be acetylated  Mimics hypo-acetylation  gene silencing o Mutate lysine to glutamine  Glutamine is uncharged AA  Mimics hyper-acetylation  gene expressio
More Less

Related notes for MIMM 323

Log In


Join OneClass

Access over 10 million pages of study
documents for 1.3 million courses.

Sign up

Join to view


By registering, I agree to the Terms and Privacy Policies
Already have an account?
Just a few more details

So we can recommend you notes for your school.

Reset Password

Please enter below the email address you registered with and we will send you a link to reset your password.

Add your courses

Get notes from the top students in your class.