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MBB267 > Mitchell > Flashcards

Mitchell Flashcards

1
Q

What does recognition of DNA seqs by DNA binding proteins allow?

A
  • targeting of RNA pol to specific genes
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2
Q

What do genes transcribed at high rate show?

A
  • high density of RNA pol along length of gene
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3
Q

Where is DNA of euk cells packaged?

A
  • chromatin
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4
Q

What is the Barr body?

A
  • eg. of densely packaged chromatin
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5
Q

Why must certain regions of chromatin be unravelled?

A
  • in order for specific genes to be transcribed

- for TFs to gain access to DNA

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6
Q

How are genes switched on/off through chromatin?

A
  • localised unfolding and repackaging of DNA into chromatin
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7
Q

Do diverse types of somatic cells have same DNA content and gene expression?

A
  • same DNA content

- differential patterns of gene expression

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8
Q

What do cells switch genes on/off in response to?

A
  • extracellular stimuli

- eg. nutrients or signalling molecules from other cells

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9
Q

What recognises signalling molecules?

A
  • receptor molecules on cell surface or localised w/in cell
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10
Q

Why are signalling pathways that stimulate cell growth and division usually activated for limited periods?

A
  • constitutive activation can cause uncontrolled growth and cancer
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11
Q

At what level are gene expression levels in euk cells mainly reg?

A
  • transcriptional level
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12
Q

How is coding pot of euk genomes increased?

A
  • through alt pre-mRNA splicing patterns that prod distinct protein products
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13
Q

What can errors in splicing patterns cause?

A
  • genetic diseases

- eg. muscular atrophy

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14
Q

When can gene expression also be reg?

A
  • post transcriptionally at level of alt splicing, mRNA translation and/or mRNA stability
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15
Q

Why are some transcripts localised to specific regions of cell?

A
  • ensure targeted expression of protein
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16
Q

What is the role of ribonucleases?

A
  • provide activities central to process functional RNAs from 1° transcripts
  • quality control systems that remove misassembled/incorrectly processed RNAs
  • timely degradation of mRNA
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17
Q

What is the GAL switch?

A
  • model inducible genetic control system in budding yeast

- monosaccharide sugar galactose must be converted to glucose for it to be metabolised by euk cells

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18
Q

Why is transcriptional control of euk genes important?

A
  • for cellular differentiation, dev and cellular signalling

- response to env

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19
Q

What does transcriptional control of euk genes involve?

A
  • cis regulatory elements and DNA binding proteins
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20
Q

How does dispersal of regulatory elements vary in humans and E. Coli?

A
  • far more dispersed than E. Coli
  • in adults = distal promoter
  • in embryonic cDNAs = proximal promoter
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21
Q

How does sex lethal gene determine sex in fruit flies?

A
  • expressed form embryonic PE promotor only in females
  • expressed from PL promoter further upstream in males and females
  • PE and PL gen distinct but overlapping transcripts
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22
Q

What is promoter region?

A
  • where RNA pol assembled and lies immediately upstream of transcrip start site
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23
Q

Where are TATA boxes found?

A
  • promoters of highly inducible genes
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24
Q

What is a TATA box?

A
  • approx 30nts 5’ of TSS
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25
Q

What are CpG islands?

A
  • clusters of dinucleotide CG w/in promoter region that are unmethylated
    • -> seq CpG usually methylated
    • -> seq CG rare as targeted by DNA methyltransferases and can mutate to TG when methylated
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26
Q

How can TSS of gene be mapped?

A
  • viral RT enzymes can gen ssDNA from RNA template
  • start DNA primers can be designed by knowing small part of seq and annealed to ssRNA
  • DNA primer extended 5’ to 3’ by RT until reaches 5’ end of RNA
  • lengths of cDNA products measured by high res PAGE and mRNA 5’ ends inferred
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27
Q

What is deletion analysis of promoter regions used for?

A
  • map reg elements w/in promoter region
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28
Q

How is deletion analysis of promoter regions carried out?

A
  • promoter region cloned upstream of coding seq of reporter gene w/ easily assayable activity (eg. lacZ) insuitable plasmid
  • transfection of cells w plasmid allows reg expression of reporter gene
  • short stretches of promoter region can be removed from either end and truncated promoter seqs reinserted into vector and tested for ability to stimulate transcrip in vivo
  • by making cell extracts from transferred cells and assaying for level of reporter gene
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29
Q

When is linker scanning mutagenesis of promoters carried out?

A
  • after promoter region defined by deletion analysis
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30
Q

How is linker scanning mutagenesis of promoters carried out?

A
  • mutate short overlapping stretches of nucleotides to gen series of constructs, each containing randomised nucleotides w/in specific region of same length of DNA
  • mutations that cause decrease in reporter protein activity define short seqs for transcriptional activity
  • typically contain TATA box and promoter-proximal regions
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31
Q

How do distant enhancer elements stimulate transcrip?

A
  • can be enhancer or silencer elements
  • typically consist of arrays of recognition sites for nt-specific DNA binding proteins
  • generally function in cell type specific manner and activate set of genes close together in chromosome
  • enhancers composed of arrays of seq elements
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32
Q

How are transcrip regulatory elements organised?

A
  • mammalian genes contain promoter-proximal elements, distal enhancers and promoter elements
  • promoter-proximal elements found w/in ≈200 nts upstream of TSS
  • enhancer regions can be 10s of hilobases upstream of TSS, w/in introns or up to 10s of kilobases downstream of coding seq
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33
Q

How are yeast genes organised?

A
  • highly compact genome w/ 1 protein encoding gene per 2kb DNA
  • promoter and terminator regions short
  • TATA box ≈90 nts upstream of TSS
  • less regulatory elements, typically reg by single upstream activating seq or upstream repression seq
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34
Q

Where is preinitiation complex (PIC) is assembled on promoters?

A
  • PIC of general TF assembled at pol II promoters
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35
Q

How is preinitiation complex assembled on promoters?

A
  • TATA box binding protein subunit of TFIID complex directly binds to TATA box seq element ≈30 nts upstream of TSS
  • TATA box binding protein binds to minor groove and gen bend in DNA
  • TFIIB complex binds to TFIID
  • RNA pol II assoc w/ TFIIF –> binds TFIID/TFIIB complex
  • TFIIE and TFIIH bind to gen PIC
  • TFIIH has DNA helicase activity, separates 2 strands allowing primer synthesis
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36
Q

What do proks and euks both have in terms of transcrip reg?

A
  • RNA pol and initiation factors that target pol to promoter regions
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37
Q

What do regulatory proteins target in euks and proks?

A
  • proks = directly target pol

- euks = impact pol indirectly via chromatin structure and mediator complex

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38
Q

What is the mediator complex (euk regulatory proteins target)?

A
  • large complex of 30+ diff proteins conserved from yeast to man
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39
Q

How is transcrip reg in euks by mediator complex and TFs?

A
  • mediator complex and general TFs allow basal level of transcrip by RNA pol II
  • specific TFs are reg proteins that stimulate or repress basal level of transcrip from certain protein
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40
Q

What is a basal level of transcrip?

A
  • nonstimulated, nonrepressed level of transcrip by RNA pol II
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41
Q

Why is linkage of functionally linked genes simpler in proks?

A
  • bacteria have operons

- genes expressed in same time in same amount

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42
Q

How is linkage of functionally linked genes by specific TFs experimentally investigated?

A
  • expression levels of gene array measured upon transcrip repression of no. of test genes
  • horizontal lines = diff groups
  • vertical lines = diff conditions
  • specific TFs 2-10% of euk proteome, but expression of most genes is reg
  • achieved as transcrip control of functionally linked genes coord by sets of specific TFs
  • -> these regions defined by transcriptional profiling and cluster analysis of global gene expression using microarray
  • compare genes and see some activated and repressed in same pattern (clustered in groups), so can identify genes coordinately expressed w/o knowing function or running other experiments
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43
Q

What is combinatorial control?

A
  • TFs function by it

- small no. factors can be arranged in v large no. combos

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44
Q

What is the structure of TFs?

A
  • modular

- typically DNA binding domain and activation/repression domain, separated by flexible linker region

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45
Q

What is the role of activation domain in TFs?

A
  • fusion of AD to other DNA binding proteins confers control of genes w/ cognate binding site
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46
Q

What assay clearly demonstrates modular structure of TFs?

A
  • yeast 2-hybrid
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47
Q

How is yeast 2-hybrid carried out?

A
  • Gal4 stimulates expression of genes w/ GAL upstream activating seq w/in promoter region in response to galactose
  • DNA binding domain and activation domain fused separately to bait and prey proteins
  • if bait and prey interact, Gal4 protein reconstituted and can promote transcrip of reporter gene containing promoter-proximal GAL upstream activating seq
  • identifies proteins that interact in vivo w/ protein of interest and to map region of interaction
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48
Q

What are typical reporter genes used in yeast 2-hybrid assays?

A
  • HIS3 = false +ves filtered out by using increased concs of comp inhibitor
  • ADE2 = coloured colonies
  • lacZ = coloured colonies
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49
Q

How do multiprotein complexes assemble on enhancers?

A
  • enhancer regions consist of multiple reg elements that bind diff TFs cooperatively
  • HMGI (DNA BP) binds to minor groove, causing it to bend
  • enhancer complex containing multiple subunits assembled through large no. protein-protein and protein-DNA interactions
  • -> multiple weak interactions between sTFs strengthened by interactions w/ DNA
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50
Q

What is the best characterised enhancer complex, and what is its structure?

A
  • virus-inducible β-interferon complex

- contains heterodimers that cooperatively bind to adj regulatory elements in HMGI presence

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51
Q

What is an example of a model genetic reg system?

A
  • yeast GAL gene switch
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52
Q

How does the yeast GAL gene switch work?

A
  • galactose –> glucose req Gal2 permease and 3 enzymes (Gal1, Gal7, Gal10)
  • expression reg in response to galactose by sTF Gal4, regulator protein Gal80 and galactose sensor Gal3
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53
Q

What are gel shift assays used for?

A
  • purify TFs that interact w/ reg element of known seq
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54
Q

How are gel shift assays carried out?

A
  • short ds DNA molecule containing reg element incubated w/ fractionated nuclear extract
  • complexes of DNA bound to protein have slower electrophoretic mobility than non bound DNA
  • allowing identification of fractions containing cognate sTF
  • reiterative cycles of fractionation based on differing physical properties
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55
Q

What question can in vitro assay of TF activity investigate?

A
  • is purified DNA binding protein actually a TF
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56
Q

How is in vitro assay of TF activity carried out?

A
  • performed in presence and absence of purified protein

- using templates that either do or don’t contain TF binding site

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57
Q

How is an in vitro (transfection) assay carried out?

A
  • combined w/ in vitro to prove in cellular context
  • plasmid bearing cloned sTF and reporter gene, w/ cognate sTF binding site, introd into cells and measure amounts of reporter mRNA/protein
  • expect increase if cloned gene encodes cognate TF
  • allows mutational analysis of sTF
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58
Q

What is the structure and role of ligand binding domains?

A
  • ligand binding domains of nuclear receptor are folded into compact structures
  • function as activation domains when bound to their hormone
  • binding triggers conformational change facilitating interaction w/ coactivators
  • eg. estrogen receptor when binds estrogen or tamoxifen
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59
Q

What are the characteristics of acidic activation domains?

A
  • largely unstructured but fold upon binding coactivators
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60
Q

How can activator/coactivator interactions be reg?

A
  • adenylate cyclase gen cAMP, which activates protein kinase A
  • sTF CREB phosphorylated by catalytic subunit of protein kinase A
  • CREB must be phosphorylated to bind coactivator, CREB BP
  • CREB binds to cAMP response elements, allowing transcrip of genes reg by them
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61
Q

What can defects in transcrip repressor domains cause?

A
  • uncontrolled growth –> cancer
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62
Q

How can transcrip repressors be identified?

A
  • genetically or biochemically
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63
Q

What is base-specific recognition of DNA binding proteins?

A
  • bind in seq specific manner
  • base recognition occurs through interactions made in major groove of DNA
  • res w/in α helices contact edges of bases
  • basic residues may also interact w/ phosphate backbone
  • bacteriophage repressors often dimers
  • recognition helix inserted into major groove and supported in helix-turn-helix fold
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64
Q

What is the homeodomain fold assoc w/ in sTFs?

A
  • morphogenesis
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65
Q

Where is the homeodomain fold found?

A
  • euk TFs

- similar to helix-turn-helix fold of bacterial repressors

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66
Q

What is the homeodomain fold made up of?

A
  • 60AAs encoded by 180 nt long homeobox DNA seq
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67
Q

What is the structure and role of Hox genes?

A
  • homeobox seqs highly conserved and readily identifiable w/ seq alignment tools
  • key role in anterior-posterior dev
  • clustered and order correlates w/ spatial and temporal expression
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68
Q

What is the most common DNA-binding fold in human cells?

A
  • zinc-finger proteins
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69
Q

Are all zinc-finger proteins involved in DNA binding?

A
  • no, some in RNA and protein interactions
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70
Q

What is the structure of zinc-finger proteins?

A
  • β, β, α protein fold centred around central Zn2+
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71
Q

How do zinc-finger protein perform their role?

A
  • helix slots into major groove and forms specific interactions w/ adj nucleotides
  • zinc-finger proteins can be designed to target specific seqs
  • Zn2+ has contacts w/ 4 AAs (2 Cys and 2 His or 4 Cys)
  • -> found as 2 closely spaced pairs of residues in 1°seq, separated by intervening seq thats looped when drawn out (= zinc ‘finger’)
  • Zn2+ stabilises 3D arrangement of α helix and β sheets
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72
Q

What are the 2 major families of zinc finger proteins?

A
  • C2H2 = contain multiple zinc fingers and bind DNA as monomers
  • C4 = in nuclear receptors, contain 2 zinc fingers and bind as dimers
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73
Q

What are basic leucine zipper proteins?

A
  • family that comprise of Leu zipper to allow dimerisation
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74
Q

What are the features of basic leucine zipper proteins that allow them to perform their role?

A
  • coiled-coil dimer linked through parallel amphipathic α helices
  • w/ Leu at every 7th position –> contributes strongly to hydrophobic interactions between 2 α helices
  • stably bound to by add ionic interactions
  • region rich in basic residues to allow DNA binding
  • extended helices grip DNA of adj major grooves
  • basic helix-loop-helix proteins structurally related to bZIP proteins but have non helical regions that connect Leu zipper region and helix involved in DNA binding
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75
Q

How do TF interactions diversify transcriptional control?

A
  • TFs typically function as homo or heterodimers
  • some heterodimers recognise same DNA seqs –> so target genes respond to distinct combos of activation domains under diff conditions
  • combo of diff TFs can gen alt heterodimers –> increase diversity of DNA seqs, so increase pot targets
  • bZIP proteins can also bind repressors, blocking DNA binding
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76
Q

How can combinatorial control also be achieved?

A
  • w/ sTFs that composite DNA seq elements
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77
Q

How does cooperative binding of distinct DNA binding proteins occur?

A
  • 2 proteins that’d bind weakly to adj sites w/in DNA in isolation may be able to form stable transcrip complex through intermolecular protein-protein interactions
  • dep upon relative position of 2 recognition elements w/in DNA
  • multiple weak interactions often underlie specificity of transcrip control
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78
Q

How is euk DNA packaged into chromatin?

A
  • DNA of interphase packaged together w/ histones, non histone proteins and RNA into chromatin
  • further compaction req to make metaphase chromosomes
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79
Q

Why must DNA be packaged?

A
  • to fit into nucleus
  • but still remain accessible at mol level for transcrip, rep and repair
  • prevents other factors from binding
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80
Q

How much of nucleus is made up of DNA?

A
  • ≈ 5% vol

- ≈ 60% vol of sperm cell

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81
Q

What are chromosome territories?

A
  • indiv chromosomes occupy own specific area of euk cell nucleus
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82
Q

What happens to chromosome territories during transcrip?

A
  • decondensed
  • actively transcribed genes found in DNA that’s looped out
  • multiple actively transcribed genes assoc w/ RNA pol and factors in transcri factories typically close to nuclear pores
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83
Q

Why does chromatin extracted from nuclei under low salt conditions resemble ‘beads on a string’?

A
  • particles are nucleosomes
  • joined together by linker DNA
  • nucleosomes consist of DNA wound around 1 of histone proteins
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84
Q

How is chromatin organised?

A
  • chromatin extracted from nuclei under low salt conditions resembles ‘beads on a string’
  • when extracted under physiological salt conditions, more condensed form, ≈ 30nm fibre
  • further packaged into higher order structure
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85
Q

What is the structure of nucleosome particle?

A
  • octameric core, w/ 2 copies of H2A, H2B, H3 and H4
  • 2 H2A/H2B and H3/H4 dimers interleaved in handshake like interaction
  • 147bp of DNA makes 1.7 left handed turns round core
  • length of linker DNA varies between 10 and ≈ 100bp
  • H1 binds DNA as it enters and exits nucleosomes
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86
Q

When are nucleosomes released?

A
  • limited nuclease digestion of chromatin
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87
Q

What is the structure of histones?

A
  • core histones one of most conserved euk proteins
  • histone variants found in some nucleosomes
  • consist of globular protein domain w/ flexible extensions at N-ter or both ter
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88
Q

How is most DNA w/in sperm packaged?

A
  • w/ other Arg ich proteins called protamines
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89
Q

What is the structure of 30nm fibre of chromatin?

A
  • in vitro, 10nm fibre condenses into “30nm fibre” at physiological salt conditions
  • packaging req histone tails and linker histone H1
  • 2 classes of model proposed for structural organisation
    • -> single helix
    • -> 2 stranded left-handed double helix of nucleosomes
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90
Q

How can core histone tails be post translationally mod?

A
  • acetylation of Lys
  • mono/dimerisation of Lys
  • mono/symmetrically dimethylation/asymmetrically dimethylation of Arg
  • phosphorylation of Ser/Thr
  • ubiquitylation of Lys
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91
Q

Are post translational mods of core histone tails mutually exclusive or dep?

A
  • can be either
  • mutually exclusive = eg. methylation of Lys blocks acetylation
  • mutually dep = eg. ubiquitination of H2B req for methylation of H3K4
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92
Q

How is the Lys side chain of histone mod?

A
  • most involve ε-amino group of Lys
  • histone acetyltransferases add acetyl groups to neutralise +ve charge
  • readily reversed by histone deacetylase complexes
  • methyl groups slowly removed by lysine-specific demethylases (allowing deacetylation to occur)
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93
Q

How are heterochromatin and euchromatin packaged in interphase cells?

A
  • heterochromatin = densely packaged

- euchromatin = loosely packaged

94
Q

What does constitutive heterochromatin contain, and how is it inherited?

A
  • repetitive DNA seqs = telomeres, centromeres and satellite DNA
  • stably inherited through cell division
95
Q

What is the structure of facultative heterochromatin?

A
  • densely packaged but able to decondense
96
Q

What is heterochromatin now used to refer to?

A
  • transcriptionally inactive DNA
97
Q

What are the 2 markers of heterochromatin?

A
  • MeH3K9 (meth H3 at position Lys 9)

- MeH3K27

98
Q

What are the 2 markers of actively transcribed DNA?

A
  • AcH3K9

- MeH3K4

99
Q

What is methylation and acetylation of core histones important for?

A
  • structural transition of chromatin between condensed and decondensed state
100
Q

Where are diffs between mod patterns of core histones found between?

A
  • actively transcribed genes and heterochromatin
101
Q

What is the histone code?

A
  • histone mods affect chromatin structure both in trans (bound by factors) and in cis (nucleosome interactions)
102
Q

What is the purpose of ChIP (chromatin immunoprecipitation)?

A
  • shows histone mod status of genes

- allows analysis of protein/DNA interactions in vivo

103
Q

How is ChIP carried out?

A
  • interactions stabilised by chemical crosslinking (covalently links DNA to protein)
  • DNA fragmented and protein/DNA complexes purified by immunoprecipitation (using diff antibodies)
  • DNA analysed by PCR or microarray
  • antibodies specific to acetylated or methylated histones and gene chips provide genome wide histone maps
104
Q

How can chromodomain bind methylated histone tails?

A
  • reading histone code req proteins that specifically recognise mod nucleosomes
    methylated H3K9 mark req for heterochromatin formation –> recognised by proteins containing chromodomain
  • heterochromatin protein I (HPI) is most abundant protein in heterochromatin –> H3 peptide binds HPI as ‘missing’ strand in structure
  • many chromodomain proteins also have adj structurally related chromoshadow domain
    –> allows interaction w/ other chromoshadow domain proteins
  • ∴ HPI can draw together adj nucleosomes carrying H3M9me3 mark
105
Q

How is heterochromatin formed by HPI and histone methyltransferases (HMTs)?

A
  • heterochromatin spreads from initiation point and extends until boundary elements (spreading req nucleosomes to be present)
  • spreading dep upon H3K9 trimethylation and req HP-I and Suv3-9
  • Suv3-9 has chromodomain –> HMT activity stimulated by interaction of chromodomain to adj H3K9me3 nucleosomes
106
Q

How does chromatin-mediated repression occur in yeast?

A
  • heterochromatin silences gene expression at rDNA locus, telomeres, centromeres and mating type locus
  • gene conversion occurs at active MAT locus upon cell division
  • repression of HMLα and HMRa loci is dep upon adj silencer regions
  • heterochromatin formation promoted through histone deacetylase activity
  • protein RAP1 binds DNA in silencer regions, then recruits Sir proteins
  • Sir2 removes acetyl groups from histones, then can bind to deacetylated histones and adj nucleosome deacetlyated
  • histone hypoacetylation allows chromatin condensation
107
Q

What is the role of chromatin boundary elements?

A
  • define edges of heterochromatic regions
108
Q

What is the role and structure of insulator elements?

A
  • prevent heterochromatin spreading into actively transcribed genes
  • block cross activation or repression enhancer/silencer elements
  • consist of clusters of binding sites for specific DNA binding proteins
109
Q

Where is the nuclear matrix found, and what is its role?

A
  • lies outside chromatin regions of nucleus

- structural framework fo rmol processes

110
Q

What is the role of matric attachment regions w/in DNA?

A
  • tether chromatin to nuclear matrix and demarcate chromatin domains
111
Q

What are polycomb protein complexes and trithorax protein complexes critical for?

A
  • polycomb = maintenance of heterochromatin of Hox genes

- trithorax = maintain Hox genes in transcriptionally active site

112
Q

What do polycomb protein complexes contain?

A
  • H3K27-specific histone methyltransferase

- enhancer of zest

113
Q

What do trithorax protein complexes contain?

A
  • contain histone methyltransferase that introd H3K4 epigenetic mark for transcriptionally active chromatin
114
Q

What domain do many histone methyltransferases contain?

A
  • catalytic SET domain
115
Q

What are hypersensitive sites (in DNA)?

A
  • rapidly degraded upon incubation w/ DNA
116
Q

How was it shown that nuclease-hypersensitive sites are in actively transcribed genes?

A
  • digestion of chromatin w/ micrococcal nuclease or DNase I can release nucleosomes
  • -> DNA assoc w/ nucleosomes protected, whereas nucleosome free DNA is nuclease snesitive
  • -> resolved through agarose gels
  • DNA hybridised to gene specific probes
  • showed that 5’ end of globin gene nuclease sensitive in cells where its actively transcribed
  • S blotting to see if particular region of DNA in nucleosome
117
Q

What do nucleosome free regions in promoters and terminators allow to be mapped?

A
  • genome wide map of nucleosome occupancy, using histone specific antibodies
118
Q

What did ChIP assays reveal about nucleosome free regions?

A
  • found in promoter and terminator regions
119
Q

Nucleosomes adj to nucleosome free regions are enriched with what?

A
  • euchromatin markers
120
Q

How do activators direct histone acetylation of promoters?

A
  • histone deacetyltransferase complex SAGA is a coactivator
  • recruited to UAS seqs through interaction w/ transcriptional coactivators, eg. Gcn4 and Gal4
  • acetylation of histones loosens nucleosome interactions and provides recognition sites for proteins w/ bromodomains
  • -> eg. chromatin remodelling machines and the general TF, TFIID
121
Q

What is the catalytic component of SAGA complex, and what is its role? (not on slides)

A
  • Gcn5
  • triggers acetylation of multiple sites w/in core histones
  • hyperacetylation leads to decondensation of nucleosome structure at level of nucleosome interactions and by recruiting other factors that promote open chromatin structure
  • proteins w/ bromodomains then able to interact w/ acetylated histones
122
Q

What is the role of chromatin remodelling complexes?

A
  • have ATP dep DNA helicase activity that can push DNA into nucleosomes, causing them to “slide” along DNA
  • bind to activation/repression domains of TFs
123
Q

How do repressors direct histone deacetylation of promoters?

A
  • Sin3/pd3 histone deacetylase complex is transcriptional repressor (Rpd3 is catalytic subunit)
  • recruited to upstream regulatory seqs of many genes through interaction w/ repression domain of Ume6 by Sin3
  • deacetylation of core histones leads to chromatin condensation and subsequent transcriptional repression
  • other corepressor complexes contain histone methyltransferases (in mammalian cells)
124
Q

How does DNA methylation cause transcriptional activation?

A
  • cells in higher euks express cytosine methyltransferases
  • DNA methylation of CpG islands correlates w/ transcriptional repression
  • m5c sites recognised by proteins such as MeCP2
  • binding of MeCP2 recruits HDAC (histone deacetylase) or HMT (histone methyltransferase) complexes
125
Q

What is position effect variegation?

A
  • repositioning normally well expressed gene close to heterochromatic gene can potentially result in its transcriptional repression
126
Q

Are genes next to heterochromatin expressed?

A
  • can be in some cells, but not others

- prod mosaic expression pathway

127
Q

What are some examples of position effect variegation?

A
  • mottled allele of white eye locus in fruit flies

- telomere position effect in budding yeast

128
Q

What is X chromosome inactivation, and how is it controlled?

A
  • random inactivation of maternal/paternal X chromosome to allow dosage compensation
  • silenced chromosome visible as Barr body
  • controlled by X inactivation centre
    –> encodes no. noncoding RNAs, inc Xist transcript
  • ## Xist retained in nucleus and binds along length of inactive X chromosome
129
Q

What is a Barr body?

A
  • how inactivated chromosome visualised
130
Q

What is Xist, and what is its role?

A
  • X inactive specific transcript
  • retained in nucleus and binds along length of inactive X chromosome
  • recruits polycomb repressor complex 2, which contains Enhancer of zest and induces heterochromatin formation
  • expression reg by other noncoding RNAs encoded in X inactivation centre, inc Tsix (its antisense transcript
131
Q

How does RNA mediated trancsrip repression occur in yeast?

A
  • H3K4me found downstream of Gal10 gene during growth in glucose
  • Gal10 noncoding RNA silences GAL expression by recruiting HDACs and HMT, leading to chromatin condensation
  • heterochromatin stably inherited, so only low levels of such noncoding RNAs req for efficient suppression
132
Q

What are unstable RNAs gen in transcrip called in yeast?

A
  • cryptic unstable transcripts
133
Q

Where are unstable RNAs gen from in euks?

A
  • nucleosome free regions in promoters of normally expressed genes
134
Q

How do euk promoters gen unstable RNAs?

A
  • transcrip from some promoters can be bidirectional, gen either protein coding transcript or unstable noncoding RNA
  • -> true of many pol II promoters in euk cells
  • -> can “toggle” in single cell, allowing rapid response to regulatory signals
135
Q

How can noncoding RNAs block TF binding?

A
  • SER3 gene in yeast req for Ser biosynthesis and tightly repressed in rich medium
  • intergenic region upstream of SER3 actively transcribed, gen noncoding RNA, SRG1
  • active transcript of SRG1 blocks binding of TFs at SER3 promoter by transcription interference mechanism
136
Q

What is RNA interference used for?

A
  • deplete specific gene products and underlies natural mechanism of gene reg
137
Q

How is RNA interference carried out?

A
  • dsRNA digested into ≈25nt fragments by endonuclease Dicer (makes staggered cuts)
  • 1 strand incorp into RNA-induced silencing complexes or RNA-induced transcriptional silencing complexes
  • complexes targeted through bping to specific mRNAs or genes, leading to mRNA degradation or transcriptional repression
138
Q

How can RNA interference be used on centromeric heterochromatin in fission yeast?

A
  • upon mitosis, phosphorylation of H3S10 causes HPI depletion
  • DNA rep dilutes histone heterochromatin marks and allows brief bidirectional transcrip pericentromeric repeats
  • RNA interference machinery gen RITS complex that recruit Suv3-9 to centromere and restore HPI binding and heterochromatin formation
139
Q

What factors of sTFs are key to expression of a given gene?

A
  • effective conc
  • availability
  • activity
140
Q

By what pathways can TF activity be regulated?

A
  • de novo synthesis = prod more of TF
  • ligand binding = prod active TF once bound to ligand
  • post translational mod = eg. phosphorylation
  • formation of protein complex = most proteins don’t function as monomers, many do at heterodimers
  • release of inhibitor molecule = disassemble protein/inhibitor complex to release active protein
  • proteolytic activation = activated through proteolytic cleavage
141
Q

How is TF activity reg?

A
  • often in response to extracellular signal
  • signalling molecules recognised by specific transmembrane cell surface receptors
  • -> signalling molecule doesn’t enter cell
  • -> induces response in receptor, triggering signal to be relayed w/in cell via other molecules
  • -> sTF activated
  • alt, small lipid soluble hormones pass through plasma membrane and bind directly to TF
142
Q

What are intracellular receptors which are also TFs?

A
  • nuclear receptors
143
Q

What is an orphan receptor?

A
  • receptor without identified ligand
144
Q

What some examples of common lipid soluble hormones, and what is their structure

A
  • cortisol, retinol, thyroxine
  • heterozytic ring structures (1 or 2 rings)
  • lipid soluble so can pass through membrane
  • soluble in cytosol
  • bind specifically to TFs (then TFs = nuclear receptors)
145
Q

What is the domain organisation of nuclear receptors?

A
  • highly conserved central C4 zinc finger binding domain
  • C-ter ligand binding domain
  • N-ter activation/repression domain –> diff variants arise through alt splicing and phosphorylation, and responsible for differential response of receptors in diff cell types
  • typically function as homomeric or heterodimeric dimers
146
Q

What do DNA binding domains have nuclear receptors contain, and what is their role?

A
  • 2 C4 zinc finger domains
  • proximal zinc finger recognises response element
  • distal zinc finger mediates dimerisation
147
Q

What is role of homomeric vs heterodimeric nuclear receptors?

A
  • homomeric (eg. estrogen receptor) bind palindromic repeats
  • heteromeric (eg. retinoic acid receptor) bind direct or inverted repeat seqs and contain common monomer, retinoid X monomer
148
Q

How are heterodimeric nuclear receptors activated to promote transcrip initiation?

A
  • localised only in nucleus
  • in absence of ligand RXR bound t corepressor
  • -> recruits HDACs and blocks transcrip
  • ligand bound to nuclear receptor displaces corepressor protein from RXR
  • heterodimeric receptor binds HAT complexes that hyperacetylate nucleosomes and interacts w/ mediator complexes
149
Q

How are homodimeric nuclear receptors activated to activate transcrip?

A
  • localised in cytp in absence of ligand
  • anchored in complexes w/ inhibitor protein = heat shock proteins
  • hormone binds receptor when enters cell
  • -> releases nuclear receptor when enters cell
  • -> ligand binding domain of homodimeric nuclear receptor contains nuclear localisation signal and is sufficient for ligand dep import into nucleus
150
Q

What is the general cell surface receptor signalling pathway?

A
  • binding of ligand to cell surface receptors triggers formation of receptor complex (typically dimerisation of receptor)
  • transduction involves activation of 1 or more protein kinases
  • receptor can directly activate TF or multiple steps
  • sometimes 2° signalling molecule gen w/in cytosol
  • TFs activated in nucleus or cyto
151
Q

Are receptor molecules specific?

A
  • no, can induce more than 1 response pathway
152
Q

What are cytokines, and what are they important for?

A
  • small secreted polypeptides

- important for cell growth and differentiation

153
Q

What are JAKs?

A
  • protein kinases assoc w/ cytokine receptors
154
Q

What happens during JAK/STAT pathway?

A
  • erythropoietin prod by kidney in response to decrease blood oxygen
  • induces RBC proliferation by stimulating expression of anti-apoptotic factor Bcl-xL by binding STAT5
  • dimerisation causes JAK2 kinases to be brought closer to phosphorylate each other
  • decreases KM, so increased kinase activity
  • SH2 STAT domain activated by JAK2
  • STAT dissociates and dimerises
  • exposing nuclear localisation signal, enabling import into nucleus
155
Q

What are STAT proteins, and what is their structure?

A
  • signal transduction and activation of transcrip

- SH2 domain, w/ ATP binding affinity

156
Q

What activity do receptor tyrosine kinases have and how is it activated?

A
  • intrinsic kinase activity
  • activated by protein hormones and cell type specific growth factors
  • interaction w/ ligand triggers receptor dimerisation, dimerised receptor autophosphorylates, activating kinase activity
157
Q

What receptor tyrosine kinases do human cells express for epidermal GF?

A
  • HER1, HER2, HER3, HER4
158
Q

How were many receptor tyrosine kinases initially identified?

A
  • cancer studies
159
Q

How is HER2 overexpression important in many breast cancers?

A
  • HER2 doesn’t directly bind ligand
  • forms heterodimeric complexes w/ HER1, 2 and 4
  • overexpression makes cells responsive to low levels of GF = constitutive growth
  • HER2 amplification in 25% breast cancers
160
Q

What is Ras, and what is its role?

A
  • small membrane bound GTPase molecular switch

- triggers kinase cascade ending in MAP kinases that enter nucleus and target sTFs

161
Q

When is Ras active/inactive?

A
  • active when bound to GTP, by GEFs

- inactive when bound to GDP, by GAPs

162
Q

Where are mutations w/ RTK/Ras/MAP kinase pathway found?

A
  • wide range of tumours
163
Q

How is the TF, CREB activated?

A
  • through cAMP/protein kinase A pathway
  • induced by GPCRs
  • binding of ligand enables receptor to function as GEF –> activates membrane anchored adenylyl cyclase via trimeric G protein
  • adenylyl cyclase converts ATP –> cAMP
  • cAMP causes release of catalytic subunit of protein kinase A
  • translocates into nucleus and activates CREB
  • phosphorylated CREB binds CREs in target genes and binds to coactivator CBP
164
Q

What are the diverse functions of RNA binding proteins?

A
  • mediate specific steps in transcrip, RNA processing, mRNA export, localisation , degradation and translation
165
Q

Do RNA binding proteins bind specifically or nonspecifically?

A
  • some bind specific seqs at low nM Kd values

- others bind nonspecifically

166
Q

How can genome wide analyses of RNA binding sites be performed at nt resolution?

A
  • iCLIP and CRAC techniques
  • assoc RNA and protein covalently crosslinked in vivo by UV irradiation
  • RBP purified using specific antibody or purification of epitope-tagged fusion protein
  • bound RNA degraded to short fragment, gen cDNA and seq using high throughput seqs
  • cross linked nts misread by reverse transcriptase and appear as seq error
  • identifying substrates of processing enzymes, so not limited to analysing stable complexes
167
Q

What are some characteristics of RNA binding proteins?

A
  • highly abundant
  • most ubiquitously expressed
  • half assoc w/ mRNA
  • NA binding domains have common ancestories, TFs have more seq homology
  • many RNA binding domains in multiple repeats or combos
168
Q

How can basic rich seqs mod RNA?

A
  • eg. RGG box
  • often regulate but not sufficient for stable binding
  • multiple low affinity reactions can cumulatively contribute to specific binding
169
Q

What is the structure of RNA recognition motif?

A
  • β sheet supported by 2 α helices
  • consensus seq w/in 2 central β-strands
  • β sheet 1° interaction for RNA recognition
  • contacts w/in loops between β-strands can confer specificity
170
Q

What is the role of RNA recognition motifs?

A
  • v high affinity for RNA
  • often 2 or more function together
  • many also medite protein interactions
171
Q

What does U1A bind to?

A
  • U1 snRNA w/ high affinity
172
Q

How does poly(A) binding protein bind poly(A) RNA?

A
  • contains 4 distinct RNA recognition motifs
  • 2 RRMs req
  • β sheets make extended binding surface
  • poly(A) chain along length of surface
  • can interact w/ 7 adenosine res (each distinctly recognised)
  • interaction characterised by extensive aromatic base and base-base stacking interactions
173
Q

Where is KH domain found?

A
  • K homology domains found in multiple copies and bind RNA or ssDNA
174
Q

What is KH domain?

A
  • ancient RNA binding domain
175
Q

What is structure of KH domain?

A
  • 2 variant forms, both w/ βααβ core
  • 2 α helices connected by loop w/ highly conserved GXXG seq and variable loop
  • nts splayed out onto hydrophobic cleft formed
  • no stacking interactions between aromatic abses and nts
176
Q

What are Sm and Lsm complexes, and what is their structure?

A
  • small proteins w/ αβ1-5 structure which forms bent 5 stranded β sheet
  • 7 proteins assemble into ring structure
  • U-rich RNA seq binds along inner ring surface
  • each nt stacked between aromatic and basic residues
  • WC functional groups recognised by specific H bonding interactions w/ Asn
177
Q

How does modular recognition of RNA by puf domains occur?

A
  • pumilio homology domains contain 8 consecutive Puf repeats
  • Puf repeats stacked giving curved structure
  • consensus aromatic, basic and acidic residues line concaved surface
  • PUM-HD binds 10nts, 8 specifically recognised by 3 AA residues in each repeat
  • few contacts between nts
  • bases stacked between side chains of residues
  • specific base recognition involves H bonding w/ key AAs
178
Q

Which proteins is ds RNA binding domain found in?

A
  • diverse range
  • inc RNase III, protein kinase R, RNA-dep adenosine deaminase
  • euk proteins can contain multiple
179
Q

What is role of ds RNA binding domain?

A
  • 65 res domain interacts w/ dsRNA in seq-indep manner, through α helices stabilised by β sheet
  • RNase III and related enzymes function in processing rRNA, tRNA and microRNAs
180
Q

What is the mechanism of splicing?

A
  • 2 transesterification reactions (so no ATP req)

- intron lariat degraded by debranching enzyme and RNases

181
Q

What is the spliceosome, and what is its role?

A
  • large dynamic complex of 5 distinct small nuclear ribonucleoproteins (snurps = U1, 2, 4, 5, 6) and other proteins
  • carries out nuclear pre-mRNA splicing
  • assembled and disassembled on pre-mRNA during each splicing event
182
Q

What do snurps contain?

A
  • 1 snRNA and 6-10 proteins
  • Sm proteins common to all pol II snurps
  • U6 bound to Lsm proteins
183
Q

What do snurps do?

A
  • bp w/ pre-mRNA and w/ each other during splicing
184
Q

How is spliceosome assembled on pre-mRNA?

A
  • bping between snRNAs and pre-mRNA, or between indiv snRNAs is key
  • U1 snRNP binds 5’ splice site
  • U2 snRNP binds branchpoint A
  • U4/U5/U6 assoc together as tri-snurp and bind to U1 and U2 snurps
  • U1, then U4 released
  • U5/U6/U2 remain in catalytic spliceosome and U6/U2 snurps catalyse splicing
185
Q

What is the spliceosome cycle?

A
  • spliceosomes undergo cycles of assembly, catalysis and disassembly
  • each step assoc w/ structural rearrangements of spliceosome complex
  • dep upon ATP hydrolysis
186
Q

Why does spliceosome cycle req ATP?

A
  • structural rearrangements req ATP to bring catalytic active form of spliceosome together
  • but chem reactions themselves don’t
187
Q

What is the role of RNA helicases in pre-mRNA splicing

A
  • ATP hydrolysis to function as chaperones to drive structural transitions
  • some function as pre-mRNA fidelity factors
  • ATP hydrolysis rates coupled to substrate discard pathways = kinetic proofreading
  • ATPase Prp28 allows assembly of U4/U5/U6 tri-snurp onto pre-mRNA
  • Snu114 and Brr2 induce U4/U6 unwinding
  • Prp2 promotes formation of active spliceosome
  • Prp16 and Prp22 induce conversion of complex C to post splicing complex and removal of spliced intron
  • other helicases promote spliceosome recycling
188
Q

How does kinetic proofreading in pre-mRNA splicing occur?

A
  • Prp16 facilitates transition between 1st and 2nd step of splicing
  • prp16 mutants w/ decreased ATP hydrolysis rates improve splicing of suboptimal substrates
  • if exons not correctly aligned after ATP hydrolysis, pre-mRNA released and degraded
  • slowed ATP hydrolysis rate enables further time for exon alignment and productive splicing
189
Q

How can mRNA splicing be analysed in vivo?

A
  • use 32P labelled RNA substrates and nuclear extracts
  • RNAs resolved by PAGE
  • looped RNA migrates slower than linear
  • substrates w/ alt seq or extracts depleted of specific proteins can be assayed
  • can determine if particular protein involved in splicing or if particular nt essential for splicing function
190
Q

What is the role of C-ter domains in RNA pol II?

A
  • C-ter domain of RNA pol II comprises multiple copies of Ser-rich heptapeptide repeat
  • C-ter domain differentially phosphorylated during transcrip
  • diff phosphorylation patterns of C-ter domain code allow recruitment of diff processing complexes at beginning (capping complex), middle (spliceosome) and end (cleavage/polyadenylation) of transcrip
191
Q

How are exon boundaries defined?

A
  • through protein interactions that bridge U1 bound at 5’ end of intron w/ U2 bound at 3’ end
192
Q

What protein-RNA and protein-protein interactions occur when splicing factors promote exon/intron recognition?

A
  • U2AF –> aids U2 snRNP binding to branchpoint
  • exonic splicing enhancers
  • SR proteins –> recognise ESEs and interact though RNA recognition motifs
193
Q

How can some exceptionally large introns be spliced?

A
  • recursive splicing mechanism

- intron removed piecemeal by regen functional 5’ splice site at spliced junction

194
Q

Why is it so important that splicing is accurate?

A
  • 1/3 human genetic diseases thought to be die to defect in pre-mRNA splicing
195
Q

What is an eg of a disease caused by inaccuracies in pre-mRNA splicing?

A
  • spinal muscular atrophy
  • in exon 7 of SMN gene
  • point mutation in exonic splicing enhancer blocks binding of SF2 and use of adjacent 3’ splice site
196
Q

What are alt splicing patterns?

A
  • each pre-mRNA can gen more than 1 coding seq
  • increasing diversity of coding pot
  • can occur in tissue specific manner, or w’in same cell as result of programmed change
  • choice of using strong canonical site or weak cryptic splice site, decided by availability of alt splicing actors (can activate or repress spicing at any given site)
  • alt splicing factors are RBPs that recognise pre-mRNA seqs knows as enhancers or silencers
197
Q

How is sex determined in Drosophila?

A
  • sex lethal (sxl) is splice site repressor that promotes own expression and that of transformer (tra) in female flies by exon exclusion
  • in early embryogenesis sxl transcribed from PE promoter in females
  • in late embryogenesis sxl trancrived from PL promoter in males and females
  • tra w/ tra2 (SR protein) activates splicing at alt 3’ splicing site in doublesex transcript
  • isoforms of dsx act as transcrip repressors for genes req for sexual differentiation in opp sex
198
Q

What is the catalytic mechanism of self splicing introns?

A
  • self splicing introns are autocatalytic ribozymes
  • group II introns splice using analogous mechanism w/ branchpoint adenosine and gen lariat intermediate
  • group I introns dep on guanosine cofactor and gen lariat intermediate
199
Q

What is the importance of group I introns having specific 3D structure?

A
  • restricted to seqs that can fold into approp structures to allow juxtaposition of splice sites and guanosine nnt
  • snRNPs mediate positioning of pre-mRNA splice sites
  • splicing apparatus facilitated evo of intorn seqs
200
Q

What is the role of nuclear pore complex?

A
  • transport of protein and RNA across nuclear membrane
201
Q

What is the structure of nuclear pore complexes?

A
  • large but contain only ≈30 diff nucleoporin proteins
  • channel of nuclear pore lined w/ EG nucleoporins (restricts passive diffusion)
  • -> larger proteins and complexes transported across pore in folded state by fd
202
Q

How is an in vivo nuclear protein import assay carried out?

A
  • import of fluorescently labelled reporter proteins into nucleus dep upon cytoplasmic cell extract
  • req presence of nuclear localisation signal
  • assaying fractionated cytosolic extracts identified key transport factors, eg. Ran
203
Q

What is Ran, and where is it found?

A
  • small GTPase
  • Ran/GEF nuclear
  • Ran/GAP is cytoplasmic
204
Q

How is Ran/GTP cycle coupled to transport?

A
  • nuclear portein transport mediated by specific carrier proteins called karyopherins
  • karyopherins can diffuse through nuclear pores and interact w/ hydrophobic FG repeat nucleoporins
  • bind tightly to Ran/GTP but only weakly to Ran/GDP
205
Q

What is the overall mechanism for nuclear import of proteins?

A
  • importins bind to their cargo proteins via NLS seqs

- binding of importin/cargo complex to Ran/GTP in nucleus causes release of cargo proteins

206
Q

What is the role of exportins?

A
  • transport proteins out of nucleus
  • bind cargo via nuclear export signals
  • bind cargo only when assoc w/ Ran/GTP
  • bound effectively in nucleus and released upon Ran/exportin dissociation in cyto
207
Q

How is the directionality of transport ensured?

A
  • differential localisation of Ran/GEF and Ran/GAP
  • affinity of karyopherins for their cargo and for GTP-bound Ran
  • differential affinity of karyopherins for their cargos upon interaction/loss of Ran binding
208
Q

How is mRNA export mediated by TAP?

A
  • mRNAs exported in Ran-indep manner that uses distinct transporter protein (=TAP)
  • TAP binds RNA and FG repeat nucleoporins
  • TAP depletion blocks mRNA export
  • mRNA binding of TAP dep upon Ref
  • binding of Ref to TAP destabilises interaction w/ RNA
  • RNA dissocites from Ref and binds TAP
  • only spliced mRNPs exported to cyto
  • TAP/mRNP complex disassembled by RNA helicase Dbp5, during mRNP remodelling
209
Q

What is Ref?

A
  • component of exon junction complex that is deposited on mRNA during splicing
210
Q

How can mRNA be localised to specific regions of cell?

A
  • random diffusion and anchoring
  • active transport
  • selective degradation
211
Q

What is a zip code binding protein?

A
  • protein which binds zip codes (= seq elements w/in mRNAs that mediate localisation )
212
Q

Where are zip code seqs?

A
  • 3’ untranslated region
213
Q

How is ASH1 mRNA localised in daughter cells?

A
  • HO endonuclease induces mating type switching in S. cerevisiae, in each round of cell division
  • switching only in mother cell as HO transcrip repressed in daughter cell
  • ASH1 localises to daughter cell during cell division
  • ASH1 mRNA localisation dep on zip code binding protein She2, She3 and Myo4
214
Q

What is the rate limiting step of translation?

A
  • initiation
215
Q

How is translational controlled globally?

A
  • mechanisms typically impact on activity of translational apparatus
  • translational efficiency of indiv mRNAs med by specific RBPs and typically involves impeding translation of that mRNA
216
Q

How is translation controlled globally by eIF2α phosphorylation?

A
  • translation globally downreg in response to cellular stress
  • stress activated protein kinases block translation by phosphorylation of α subunit of eIF2
  • phosphorylated eIF2 functions as comp inhibiot of eIF2 by binding its GEF, eIF2B
  • mRNAs encoding some stress induced proteins more efficiently translated at lower eIF2 levels
217
Q

How do eIF4E/eIF4G/PABP interactions stimulate translation?

A
  • mRNA caps and poly(A) tails recognised and bound by cap-binding complex (eIF4E/eIF4A/eIF4G) and poly(A) BP, respectively
  • eIF4G/PABP interaction circularises mRNA and stimulates translation
  • 4EBPs disrupt eIF4E/eIF4G interaction and block translation
218
Q

How is translation of 5’ TOP mRNAs reg by mTOR?

A
  • 4EBPs are major target s of protein kinase mTOR
  • mTOR inhibited by rapamycin
  • target mRNAs contain 5’ terminal oligopyrimidine motif
  • mTOR pathway stimulates translation of 5’ TOP mRNAs by phosphorylating 4EBPs, inactivating them
219
Q

What is the role of mTOR?

A
  • involved in several signalling pathways that promote cell growth and proliferation
  • 1 target of mTOR signalling is translational apparatus
220
Q

How is translation controlled by makin maternal mRNAs?

A
  • mRNAs w’in oocytes maintained in translationally inactive form in cyto and activated by cytoplasmic polyadenylation
  • translational reg of masked mRNP important in early metazoan oocyte dev
  • in inactive site, maskin blocks binding between eIF4E and eIF4G
  • stored mRNPs contain cytoplasmic polyadenylation element, w/in 3’ untranslated regions, bound by CPE BP, and maskin
  • upon fertilisation, phosphorylation of CPE BP causes dissociation of maskin and deadenylase, allowing extension of poly(A) tail and circularisation of mRNP particle
221
Q

How is translation controlled by ribosome recruitment?

A
  • many ribosomal proteins in E. COli autoreg own expression
  • mRNA and rRNA binding sites have similar structures
  • binding to mRNA blocks ribosome binding
  • expression of mammalian ferritin reg by Fe regulatory proteins
  • Fe response element binds to 5’ leader and blocks binding of 43S preinitiation complex to cap binding complex
222
Q

How does sxl repression translation of msl-2 occur?

A
  • male sex lethal 2 (msl-2) expressed in male flies and is component of dosage compensation complex
  • sxl protein binds to 3’ untranslated region of msl-2 and blocks interaction of 43S preinitiation complex w/ cap binding complex
  • for transcripts that bypass this reg, sxl also functions as failsafe mechanism –> binds to 5’ leader seq of msl-2 mRNA and blocks scanning ribosome
223
Q

Why do mRNA poly(A) tails decrease in length over time?

A
  • slowly shortened in cyto by deadenylases

- deadenylated mRNAs raipdly degraded

224
Q

What is the lifetime of mammalian mRNAs?

A
  • varies from less than 10 mins to oveer 9 hours
225
Q

How are mRNA stability and translational efficiency related?

A
  • inversely correlated
226
Q

What is the euk mRNA turnover pathway?

A
  • after deadenylation, m7G cap structure removed by action of heterodimeric capping enzyme
  • alt, deadenylated transcripts can be degraded by 3’ –> 5’ exoribonucleases
  • deadenylation and decapping are rate-limiting steps in mRNA decay
227
Q

How does mRNA stability affect expression level?

A
  • expression level of mRNA reflects rate of prod and turnover
  • so stable mRNAs expressed at higher levels than unstable if under same transcrip control
  • upon transcrip induction/repression, unstable mRNA achieves new steady state more rapidly than stable
  • changes in mRNA stability cause rapid change in steady state levels in transcrip rate remains constant
228
Q

How is mRNA turnover reg by A/U rich element (ARE)-mediated decay

A
  • unstable mRNAs often contains AREs
  • typical AREs contain multiple AUUUA motifs w/in U rich region
  • ARE mediated decay reg +vely and -vely by relative expression of ARE BPs (AUF1 and HuR respectively)
  • AUF1 and HuR reg by alt splicing and post translational mod
229
Q

How are mRNAs w/ early stop codons rapidly degraded?

A
  • by nonsense mediated decay pathway
  • dep upon mRNA being actively transcribed
  • 3’ UTR region of mRNAs assoc w/ proteins
  • -> this 3’ mRNP sensed by ribosome at termination codon
  • NMD triggered in absence of interactions between ribosome and mRNP 3’ domain
230
Q

Why are truncated proteins not commonly expressed in vivo?

A
  • most wont fold into stable structure
  • degraded by proteasome complex
  • euk cells have quality control systems to eradicate mRNAs that contain early stop codons
231
Q

What is the general pathway for expression of a euk gene?

A
  • reg of expression pathway allows change in amount of product prod
  • reg occurs at rate limiting steps in pathway
  • most genes reg primarily at level of transcrip
  • post transcriptional reg can occur through alt splicing, localisation, translational control or mRNA degradation

MBB267 (5 decks)

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