MolBio9-10 - 52 Flashcards
What are the 6 points of protein expression control?
Transcriptional control, processing control, RNA transport and localisation, translation control, mRNA degradation control, protein activity control
What is the primary purpose of gene control?
The execution of precise developmental descisions so that the proper gene is expressed in the proper cell at a proper time
What is the main mode of transcriptional control?
Transcriptional initiation control
How do transcriptional activator exert their effect?
Either directly, by recruiting one of the components of the transcriptional machinery, or indirectly, by modulating the chromatin structure
What are transcriptional activators and repressors?
Modular proteins containing separate DNA-binding and activation/repression domains
What are the 4 most common DNA binding motifs?
Homeodomain, Zn finger, leucine-zipper and bHLH
What do cooperative binding and combinatorial control achieve?
Transcriptional efficiency, specificity and yet diversity of responses
Describe transcriptional activators
Modular proteins with an activation domain and a DNA-binding domain
Give an example of a homeodomain protein
Hox
Describe the homeodomain protein
60 aminoacid domain, 3 helices
Give an example of a zinc finger protein
Ci (in Hh signalling)
Describe the zinc finger
23-26 aminoacid motif (C2H2), 2 cys and 2 his bind to Zn
Give an example of a leucine zipper
Fos
Describe the leucine zipper
Leu separated by 7aa in a helix, two major grooves separated by 1/2 turn
Give an example of a basic helix-loop-helix protein
MyoD
What are the two activation effects?
Cooperative binding - increased affinity of RNA polymerase for promoter; allostery - transition from closed to open complex
What is the advantage of cooperativity?
Greater specificity
What are the four methods of repression?
Competition, inhibition, direct, and indirect
What is EMSA?
Electrophoretic mobility shift assay
Outline EMSA
Tagged DNA, probe washed through DNA that will bind to regulatory sequences, gel retardation to find where bound
What is ChIP?
Chromatin immunoprecipitation assay
Outline ChIP
Antibody for specific activator/repressor added in to in vivo sample, and DNA that has formed a complex with that activator/repressor will be precipitated out, and the sequence subsequently identified
What does epigenetic mean?
Systems that sit on top of genetic mechanisms
What is special about epigenetic alterations to the genome?
Erased in germ cells
Where are nucleosomal modifications made?
Termini of lysine, arginine and serine side chains
What is a condition of nucleosomal modifications?
Mutually exclusive of one another
What are the main 3 nucleosomal modifications?
Acetylation, methylation and phosphorylation
What are the main two nucleosomal modification enzymes?
Acetyltransferase and methyltransferase
What to acetyltransferases do?
Indeterminately acetylate any lysine residue
What do methyltransferases do?
Very specifically methylate specific residues
What does acetylation of core hostones do?
Creates binding sites for transcriptional activation factors that contain a bromodomain
What does methylation of core histones do?
Creates binding sites for transcriptional repressors that contain a chromodomain, or activators that contain a PHD zinc finger domain
What reads the histone modifications?
Histone Code Reader proteins
What is the function of histone code reader proteins?
To facilitate attachment to other components in nucleus, leading to gene expression, gene silencing, or other biological functions
What is the relationship between histone code readers and code writers?
They can recruit each other, helping spread histone code in chromatin, which impacts on gene expression
In what 4 ways do transcription activator proteins work?
Selective nucleosome remodelling, selective histone removal, selective histone replacement or selectin histone modification
What does PRC stand for?
Polycomb Repressive Complexes
What are PRCs?
Histone Code Writers/Readers that can generate or recognise repressive chromatin modifications
Name one example of repressive chromatin modification
H3-K27 methylation by Ezh2 of PRC2
Name one example of respressive chromatin modification identification
Pc chromodomain identification and subsequent recruitment of PRC1
What is the relationship between DNA methylation and repressive histone methylation?
Close functionally - transcriptionally inactive promoters are frequently rich in methylated CpG dinucleotides
Why is mammalian X-chromosome inactivation important?
Equalizes the levels of X-chromosome derived gene products in males and females
Outline mammalian X-chromosome inactivation
Essentially dosage compensation: males have XY so only 1 dose; females have XX so 2 doses. One is always silent and once is transcriptionally active, the choice is random, and occurs early embryogenesis, and is then clonally inherited
Give a visual example of mammalian X-chromosome inactivation
Tortoise shell cats - XO orange; Xo black. Males are orange or black, females are orange, black or calico. Calico is patchy because of random X-inactivation during early embryogenesis
Outline the mammalian X-chromomsome inactivation mechanism
Involves synthesis of a non-coding RNA (Xist) from the X-inactivation centre, which binds to the chromsome in cis and promotes chromatin condensation by recruiting PRC group components
What is the Barr body?
Highly condensed inactive X chromosome at the preiphery of the nucleus of female somatic cells
What is position effect variegation?
Relocation of two functionally distinct regions of genes - euchromatin and heterochromatin
Describe euchromatin
Transcriptionally active genes
Describe heterochromatin
Transcriptionally silent genes
What does abnormal reanets of euchromatin and heterochromatin cause?
Position effects that affect the transcriptional activity of the euchromatic genes
What can aberrant chromsomal rearrangements cause?
If heterochromatin is placed next to euchromatin, it can shut down gene activity - the dominant effect is from the heterochromatin to the euchromatin, effectively spreading the silence
Is position effect variegation complete?
No - some transcriptional activity of the euchromatin usually remains
