Chromatin and Control of Gene Expression

exons

segment of DNA that codes for a specific amino acid

transcription

DNA copied into mRNA with the aid of RNA polymerase

RNA polymerase will bind to promoters that act as signals in the DNA sequence to make RNA

regulatory proteins

bind to DNA to either block or stimulate transcription, depending on how they interact with RNA polymerase

gene expression controlled by these binding to specific DNA sequences, they gain access to bases of DNA at the major grooves and possess DNA-binding motifs

eukaryotic cells regulate gene expression to maintain _______ in the organism

homeostasis

controlling gene expression often accomplished by

controlling transcription initiation

DNA-binding motifs

regions of regulatory proteins which bind to DNA

examples: helix-turn-helix, homeodomain, zinc finger (most common), leucine zipper

players in transcription regulation

DNA-binding transcription factors (upstream factors), chromatin regulators, coactivators and corepressors (mediators), basal machinery (RNA Pol II, GTFs)

main steps of transcription

1. polymerase binds to promoter sequence in duplex DNA, closed complex

2. polymerase melts duplex DNA near transcription start site, forming a transcription bubble, open complex

3. polymerase catalyzes phosphodiester linkages of two initial rNTPs

4. polymerase advances 3’ to 5’ down template strand, melting duplex DNA and adding rNTPs to growing RNA

5. at transcription stop site, polymerase releases completed RNA and dissociates from DNA

transcription initiation steps

promoter (start site) recognition

promotor binding

promoter melting

transcript initiation

promoter escape/clearance

transcript elongation

general transcription factors

required for transcription initiation- bind to promoter region of the gene

required for proper binding of RNA polymerase to the DNA

specific transcription factors

increase transcription in certain cells or in response to signals

what binds to promoter after general transcription factor?

RNA polymerase II, begins transcription at the start site

it is only when this happens first that RNA polymerase is placed in an orientation that allows for initiation of transcription

enhancers

DNA sequences to which specific transcription factors (activators) bind to increase the rate of transcription

coactivators and mediators

also required for the function of transcription factors

bind to transcription factors and bind to other parts of the transcription apparatus

2 general ways to alter phenotypes

altered structure and altered expression

altered structure

genetic variation in expressed sequence

normal expression

leads to altered mRNA, altered protein, normal levels→altered phenotype

altered expression

genetic variation in regulatory sequence

normal structure

leads to normal mRNA, normal protein, altered levels → altered phenotype

changing exon sequence (e.g. one nucleotide) can lead to

prevention of transcription (altered expression level), prevention or incorrect processing of mRNA (can’t make a protein, altered expression or structure), protein with reduced/absent function or different function (altered protein structure)

mutations

changes in the DNA sequence passed on to future generations

point mutations

can be silent, nonsense, missense

a single base substitution

single nucleotide polymorphism (SNPs) occur commonly within a population

frame-shift mutation

modification of the reading frame after a deletion or insertion, resulting in all codons down stream being different (i.e. the codon sequence is shifted)

substitution disease example

sickle cell anemia

insertion mutation disease

Huntington’s disease

deletion mutation disease

Tay-Sachs disease

PKU

insertion of premature stop codon

autosomal recessive gene mutation

rare disease

can’t convert phenylalanine to tyrosine, which is precursor for dopamine and norepinephrine

phenylalanine can build to toxic levels, major developmental effects

but if on special diet with artificial protein substitutes, developmental effects very minimized

nucleosomes

repeat unit of chromatin

block RNA polymerase II from gaining access to promoters (need to decondense DNA to open)

Swi/Snf complex

removes nucleosomes and deposits histone variants (H2AZ for example) for specialized functions (e.g. heterochromatin)

different ways to open chromatin

via modification of histone tails (acetylation and methylation)

via nucleosome mobilization

chromatin modification of histone tails

acetylation and methylation

conducted by HAT and HMT activity

bases for the histone code hypothesis

chromatin modification via nucleosome mobilization

ATP-dependent process

positioning of nucleosomes creates promoters with different requirement for remodeling

histones

small proteins containing a high proportion of positively charged (basic) amino acids (e.g. arginine and lysine) that facilitate binding negatively charged (acidic) DNA molecule

5 major types which are very similar among different species of eukaryotes

the most universal proteins in nature

among the most highly conserved genes in evolution

conserved nucleosome and histone-like structures in plants and animals

5 major types of histones

H1, H2A, H2B, H3, H4

histone folds

three-helix core domain

forms a handshake arrangement

histone tails

disordered n-terminal and/or c-terminal tails that protrude from the nucleosome through minor-groove channels

ideal located for covalent modifications

octamer

(H2A, H2B, H3, H4)2

assembly: H3-H4 tetramer → +2 H2A-H2B dimers

crystal structure of the nucleosome core particle

2.8 angstrom resolution

146 bp of DNA wrapped around a histone octamer core

DNA wrapped around so it forms 1.7 turns of a left-handed superhelix within this