Gene Regulation in Eukaryotes

Human genome contains few protein-coding genes, compared to simpler organisms = 1 gene many not just code for 1 protein

Eukaryotes are multicellular - different cell types in organism

Different sets of genes expressed so cells have mechanisms to switch genes on/off

Human genome = 3 billion bp - lot of exons

1bp = 0.34nm long

Haploid genome = ~1m

1 human chromosome = 10-73mm DNA - condensed to fit in 10um cell nucleus

Chromatin used to pack nuclear DNA

Used to condense chromatin in cell nucleus

Proteins - 8 histone cores shaped like hockey puck (11nm wide/6nm high):

• 2 x H2A
• 2 x H2B
• 2 x H3
• 2 x H4

147bp wrap around core - 1.7 left handed turns

Linker DNA (15-55bp) - bound to H1 and connects to other cores/helps packing of fibres

The core histones have N-terminal tails - modified by phosphorylation, acetylation, methylation etc

• Regulates gene expression - if packed too tightly factors can't intertogate and transcription silenced
• Helps with further condensing in mitosis

Epigenic effects - structure changes that allow access etc.

• Acetylation of Lys or Arg on histone tail = neutralises positive charges
• Phosphorylation introduces -ve charges on Ser
• Methylation of Lys (proteins) or cytosine (DNA) - silencing of transcription

• Transcription initiation
• Transcription of pre-mRNA from DNA
• Splicing = mRNA
• Processing by 5' and 3' modifications
• Translational control mechanisms
• Export from nucleus
• Translation in cytoplasm - requires control mechanisms
• mRNA decays after translation
• Post-translational modifications of proteins

DNA in eukaryotes stored in intact double helix - hard to penetrate and 'scan'

= RNAPs interact with zipped-up DNA before unwinding

• Base pairs appear chemically different - recognisable in major groove
• DNA binding protein e.g. Asp/Arg on RNAP distinguish bases and bind to them

• G - H-bond acceptor
• C - hydrogen atom
• T - methyl group
• A - H-bond acceptor

Eukaryotes have no operons - genes individually transcribed on different chromosomes
Indirect coordinated control occurs

3 RNA polymerases for transcription - polymerase II transcribes mRNA, located in nucleoplasm

RNAP II promoters - number of elements to help recruit basal transcription factors (mutations of promoters cause loss of transcription)

For RNAP II bind to DNA - TATA binding proteins (TBPs) and accessory factors bind to TATA box to form TFIID complex = recruitment of additional transcription factors -> recruitment of RNAP II

Promoters immediately upstream of transcription start site, contain dispersed sequence elements that bind transcription factors

Enhancers (stimulate promoter activity), contains closely arranged sequence elements that bind transciption factors, - may be distal

Promoters and enhancers contain sequence motifs to which transcription factors can bind
Different combinations allow varied control

Activators can recruite histone acetyl transferase (HAT) - acetylates histone tails = increased mobility of nucleosomes:

• Looser chromatin structure = promoter region more accessible
• TFIID binds more strongly to acetylated nucleosomes

Activators can also recruite chromatin remodelling complex - increased motility of nucleosomes = promoter region more accessible

BOTH facilitate binding of further transcription factors and polymerase II machinery

Gal1 - enzyme galactokinase (galactose metabolism in yeast)
Activated by Gal4 molecule - 2 seperate domains:

• DNA binding capability
• Transcriptional activation

UAS (upstream binding sequence) - 4 binding sites for Gal4 bind to and turn on gene

Gal4 manipulated - detmines if activation domain (AD) necessary and sufficient for activation of transcription

• Intact Gal4 - binds to Gal4 site = expression
• Gal4 w/out AD - binds to Gal4 site = no expression
• LexA w/out AD - binds to LexA site = no expression
• Gal4 AD/LexA binding domain fusion - binds to LexA site = expression

Transcription factor - any of various proteins that bind to DNA and play a role in the regulation of gene expression by promoting transcription

DNA binding domain = 20 contacts w/ DNA target sites

Conctacts = strong, specific binding of transcription factors to target site

Transcription factors classed by DNA binding domains:

• Homeodomain
• Zinc finger
• Leucine zipper
• Helix-Loop-Helix (HTH)

DNA binding = N-terminal long a-helix contacts major groove of DNA and reads it

Dimerisation = Consists of a short a-helix and long a-helix combined

Examples - MyoD, E12

DNA binding = N-terminal part of long a-helix contacts major groove and reads DNA

Dimerisation = C-terminal lecuine zipper domain (helix where every 7th amino acid leucine)

Examples - c-Jun, Fos, ATF

c-Jun:

• Every a-helix turn 3.6 amino acids
• Leucine side chains stick out on same side every other turn
• Two helices = hydrophobic interactions in zip-like manner
• Zippers have different dimerisation combinations - homodimers (same binding site) or heterodimers (different binding site)
• Different predicted binding patterns for each individual one

Helix 2 and 3 = helix-turn-helix structure

Helix 3 = sequence-specific interactions with major DNA groove

b-sheet and a-helix

Two cystines and 2 histidines complex zinc atom

a-helix contacts DNA in major groove

Clustered Control Regions

Each gene has local regulatory elements

Also, distant locus control region/global control region required for orderly expression = regulate batteries of genes

Direct interaction - facilitates cooperative binding to DNA

Example - Transcription during hypoxia

• Normal O2 conditions - HIF1a destroyed, cannot partner to HLF1b
• Low O2 conditions - heterodimer forms and activate transcription at genes w/ Hypoxic Response Element in promoters

Indirect interaction - via mediators, facilitates cooperative binding to DNA

Loosening of DNA binding to nucleosomes - A binds to site, causing partial unwinding of DNA from nucleosome = site B accessible

Chromatin remodelling - A binds and recruits chromatin remodeller = binding site for B accessible

Enhanceosome - higher order protein complex assembled at enhancer and regulates expression at target gene

Combination Control

Gene receives several signals each represented by 1 transcription factor (may control number of genes). Each genes needs multiple TFs to turn on gene

Signalling Pathways:

Jak/Stat Pathway:

• Cytrokine binds to receptor = receptor phosphorylated by JAK
• Stat binds to receptor and phosphorylated
• Phosphorylated stat dimer translocates to nucleus, activates target genes

Ras Pathway:

• Ligand binds to Trk which autophosphorylates
• Grb and SOS activate small GTPase Ras
• Ras triggers MAP kinase cascade
• MAP kinase phosphorylates transcription factors

Combination Control

Gene receives several signals each represented by 1 transcription factor (may control number of genes). Each genes needs multiple TFs to turn on gene

Signalling Pathways:

Jak/Stat Pathway:

• Cytrokine binds to receptor = receptor phosphorylated by JAK
• Stat binds to receptor and phosphorylated
• Phosphorylated stat dimer translocates to nucleus, activates target genes

Ras Pathway:

• Ligand binds to Trk which autophosphorylates
• Grb and SOS activate small GTPase Ras
• Ras triggers MAP kinase cascade
• MAP kinase phosphorylates transcription factors

Repression:

• Insulators
• Competition
• Inhibition
• Direct repression
• Indirect repression
• Gene silencing
• Maintenance of DNA methylation

Insulators restrict activation by enhancers by abolishing expression, blocking transcription from occuring.

Competition - repressor binds to same or overlapping DNA region as activator (mol. with highest affinity/highest concentration will win)

Inhibition - repressor binds next to activator and interferes with activation

Example - Gal1 repression by inactive Gal4

• Absense of galactose - Gal80 masks Gal4 activation domain
• Masked, inactive Gal4 binds to same site as active Gal1
• Represses Gal1 by competition

Direct - repressor inhibits transcription initiatation by interaction w/ transcription machinery

Indirect - repressor recruits factors making chromatin less accessible for transcription e.g. HDAC - histone deactylation or histone methylation/nuclesome modelling = blocks promoter sites

Example - Gal1 repression by Mig1

• Gal genes metabolise galactose
• Presence of glucose activates Mig1 TF
• Mig1 recruits Tup1
• Tup1 recruits HDACs - Indirect repression
• Tup1 disrupts transcriptional machinery - direct repression

Gene switched off by absence of activators and lack of transcription machinery recruitment

Off state reinforced by DNA methylation - methyl-C binding proteins bound - recruit HDACs and chromatin remodelling complexes = indirect and direct repression

Example - Imprinting

• Interaction of lg2 growth factor gene w/ enhancer regulated by methylation of imprinting control region (ICR)
• Maternal allele - insulator active, only H19 expressed
• Paternal allele - insulator and H19 methylated, inactive, lgf2 expressed

DNA methylation allows transmission of epigenic information (modification of gene expression) from mother to daughter cells - silencing inherited

Fully methylated CpG forms 2 hemi-methylated CpG

Hemi-methylated CpG forms fully methylated CpG

Enzyme for maintenance of methylation - DNA methyltransferase called maintenance Dnmt

Transcription begins at promoters

Genes have alternative promoters - 1000s of bp apart

Alternative promoters tissue-specific or specifically timed during development

Example - tumour suppressor gene p53 -

• Transcribed from alternative promoters
• p53 internal promoter found in 24/30 breast tumour
• Not in normal breast tissue

Production of mature mRNA in eukaryotes - ~3 RNA processing mechanisms

• Capping
• Polyadenylation
• Splicing

Transcription and processing occur in nucleus - coupled events

Capping, splicing, polyadenylation occur while RNA pol II actively enlongating RNA chain

Processing complete = mRNA transported to cytoplasm = protein translation

5' end of pre-mRNA 'capped' 20-40nt after synthesis

• Terminal phosphate at 5' end of RNA cleaved
• 5' terminal G added (7-mG) in reverse orientation
• G connected to 5' end of RNA by 5' to 5' triphosphate bridge
• This methylated at 7 position w/ purine ring 
• = m7G (7-methylguanosine cap) 

m7G protects from degredation and increases translational efficiency 

Length of cap depends how long its used for - longer caps = mRNA last longer

Polyadenylation signal (AAUAAA) recognised and mRNA cleaved 11-30nt downstream

Multiple (>200) As added at 3' end

mRNA stability and efficiency of translation

mRNA - introns need to be removed to make mature mRNA

Intron - non-coding DNA stretch (Bigger gene = more introns)

Introns are removed from between exons by splicing and exons are ligated = mature mRNA

Showing introns:

• Templace strand of DNA and mRNA heated separately
• Mixed and cooled = base pair formation
• Electron micrograph - several regions where genomic DNA and mRNA do not bp = introns

Pre-mRNA splicing occurs in nucleus and carried out by snRNAs U1 to U6 

snRNAs biind to proteins = snRNP and multiple snRNPs = spliceosome

Introns removed in 2-step transesterification reactions

• Boundaries of introns and exons identified by presence of specific nucleotide sequences
• Introns
  • Conserved 5' donor site
  • 3' acceptor site
  • Internal branch site
• U1 binds to donor site
• U2 binds to branch sequence
• U4, U5, U6 form spliceosome
• Branch point brought close to donor site = lariat
• Intron excises
• Exons ligated together

Alternative splicing occurs in formation of mRNA - different potential exons spliced out = different combinations = many different proteins translated from same DNA piece (protein isoforms)

Increases protein diverstiy and different isoforms expressed at different times in development

Choice of exon regulated in tissue or developmental-stage specific manner. Control of a. splicing:

• Negative
  • Defult, intron spliced out
  • Repressor protein may bind to pre-mRNA
  • Block access of SNPs
  • = Intron retained
• Positive
  • Default, intron retained
  • Activator protein may bind to pre-mRNA
  • Recruit splicing machinery
  • = Intron spliced out
  • Binding sites for activators may be remote from splice site (splicing enhancer)

• A cap and polyA tail
• Untranslated regions (UTRs) at 5' and 3' ends
• Open reading frame (ORF) containing triplet code - assembling polypeptide
• ORF begins w/ initiation codon and ends w/ 1 of 3 termination codons

Stops protein production from occuring

Gradual polyA shortening occurs

Rapid degredation kicks in below critical polyA length = times degredation of mRNA

50-150 nucleotide elements in 3' untranslated region of short-lived mammalian mRNAs

Typified by presence of single/tandem repeats of AUUA pentamers

Presence of elements control translational efficiency as well as:

• Deadenylation
• Decapping
• 3'-5' decay
• 5'-3' decay
• Cap-scavenging

Regulatory effects mediated by binding proteins

Small regulatory RNAs ~22bps long

Transcribed as larger precursors and processed to mature active form

Targeted to 3' untranslated regions

• If inperfect match - translation is downregulated
• If perfect match - mRNA cleaved