Week 1 - Transcription PDF

Transcription Regulated Gene Expression Not all ~22,000 genes are expressed in each cell Temporal and tissue specific expression is important for di;erentiation and function ~20-40% of genome is regulatory sequences Common genes known as constitutiv...

Transcription Regulated Gene Expression Not all ~22,000 genes are expressed in each cell Temporal and tissue specific expression is important for di;erentiation and function ~20-40% of genome is regulatory sequences Common genes known as constitutive Crucial in determining cell identity, function and response to signals Mis regulation is associated in many diseases Includes cancer RNA Polymerases Conserved across 3 domains of life Transcription Produces RNA complementary to one strand of DNA RNA polymerase separates two strands of DNA in a transient "bubble" Does not require a helicase Bubble = 12-14 bp RNA-DNA hybrid within = ~8-9 bp 3'-5' strand as template for 5'-3' RNA Transcription is pervasive Most of the genome is transcribed Even if little produces protein coding RNA Non-coding DNA sequences (98% of genome) Often transcribed into RNA molecules with important biological functions microRNA, lincRNA, rRNA, tRNA etc Transcription initiation Requires may proteins and complex cis-regulatory DNA elements RNA Polymerase I Transcribes ribosomal RNA genes 5.8S, 18S, and 28S rRNA RNA Polymerase II Transcribes all mRNAs in cell Also transcribes other RNAs  snoRNA, miRNA, siRNA, IncRNA, most snRNA Complex of 10 di;erent protein subunits Active site is at interface between 2 biggest subunits RPB1 & RPB2 Nucleotides continuously added to 3’ end of RNA Forms DNA-RNA hybrid Exit groove Where RNA leaves RNA polymerase II after being split from DNA Intake hole Nucleotides for RNA enter through pore RNA Polymerase III tRNA genes, 5S rRNA Some snRNA Genes for other small RNAs Core promoter elements Contains short (consensus) sequences as binding sites for GTFs Element Consensus General Transcription Sequence Factor BRE G/C G/C G/A C G C C TFIIB TATA T A T A A/T A A/T TBP INR C/T C/T A N T/A C/T TFIID C/T DPE A/G G A/T C G T G TFIID Determines start site for transcription Basal levels of transcription initiation Located immediately upstream (5’) of transcription start site Fixed direction/orientation relative to gene General transcription factors (GTF) Help RNA polymerase recognise promoters TFIID 12 subunits 1 called TBP ~11 additional called TAFs TBP-associated factors TATA binding protein (TBP) Subunit of TFIID Recognises TATA box Short sequence of DNA rich in A & T Signals start of gene ~30bp from start of gene Binds to DNA with 8-stranded beta sheet Rests on top of DNA like a saddle 2 protein loops droop down the sides Induces kink in DNA backbone  Bends DNA by nearly 90o TFIIB 1 subunit Recognises BRE element (TFIIB-response element) Recruited to BRE by TFIID Accurately positions RNA polymerase TFIIF 3 subunit Often recruited along with RNA polymerase Stabilises RNA polymerase interaction with TBP andTFIIB Helps attract TFIIE and TFIIH TFIIE 2 subunits Attracts and regulates TFIIH Stabilises transcription bubble By interacting with non-transcription strand to prevent collapse TFIIH 9 subunits Unwinds DNA at transcription start point Phosphorylates Ser5 of RNA polymerase CTD Prevents it from interacting with other general transcription factors Releases RNA polymerase from primer CTD of RNA polymerase II Factors bound to CTD can immediately interact with produced RNA MmRNA processing Capping, splicing and 3’ end formation Associated with C-terminal domain (CTD) of RNA polymerase II DNA damage repair DNA Helicase subunits of TFIIH have a role in nucleotide excision repair Nuclear architecture Nuclear matrix DNA replication Transcription factor binding sites near origins of replication Actively transcribed genes are replicated early in S phase Regulation of transcription Regulated by sequence-specific transcription factors Bind at cis-regulatory sequence Can interact from nearby or from a distance Enhancers or silencers Modulate (up or down) levels of initiation Orientation independent  Location variable/flexible Can be 1000s of kb away from promoter Binding sites for multiple transcription factors Transcriptional activators Transcriptional activators Functions Co-activators Co-repressors General transcriptional machinery Multiple protein domains Activation domain Recruitment of general transcription factors Regulatory domain Can mediate dimerization between factors Homo or hetero Nuclear transport Brings transcription factor to the nucleus Auto-inhibition DNA binding domain Sequence specific recognition of DNA promoter Specific amino acids can interact with di;erent bases Combinatorial control Transcription factors are often combined and recognise longer sequences Increases specificity greatly Can be homodimers or heterodimers Many binding sites tend to be palindromic Inhibitory factors can also dimerise with transcription factors Prevents factor from binding to DNA Some transcription factors have di;erent recognised sequences but bind weakly May work together to increase one another's a;inity Enhanceosome Multitude of transcription factors assembling into a macromolecular complex at enhancer sequences Transcription activators work synergistically  Summary Transcriptional activators work cooperatively Enhancers work from a distance to modulate the assembly of transcription machinery at promoter Through DNA looping Modern Model In addition to looping Proteins appear to hold loops in place Cohesins and CTCFs Get exclusion of water and it is just a mass of proteins interacting with one another Chromatin Remodellers Recruited by transcription factors Used to initiate nucleosome sliding to move histones Histone chaperones May remove histones to disassemble nucleosomes Histone-modifying enzyme Experiments indicate increase in activator expression induce large scale chromatin unfolding Regulation of Transcription factors Brought into play by extracellular signals Examples of factor activation Protein synthesis Ligand binding Covalent modification May be result of kinase cascade and ends in phosphorylation Addition of second subunit Unmasking By a chaperone etc. May keep protein in cytoplasm for example Stimulation of nuclear entry  Release from membrane Dysregulation examples in disease Upregulation Amplification Gain of function Pathway overreaction Downregulation Loss of function Overactivation of repressors Changes in target genes Chromatin architecture shifts Gene translocations Fusion transcription factors Types of Transcription Factor Domains Homeodomains Found in many transcription-regulatory proteins Mediate binding to DNA Consist of 3 overlapping alpha-helices Packed together by hydrophobic forces DNA-binding element Helix 2 & helix 3 Helix-turn-helix motif Amino acids in recognition helix make contacts with bases in major groove of DNA 3 side chains make H-bond connections with bases Arginine in flexible loop of protein contacts bases in minor groove Leucine-Zipper domains Consist of 2 long, intertwined alpha-helices Hydrophobic side chains stretch out into space shared between them Many side chains are leucines Tightly packed = very stable Extensions from helices straddle the DNA major groove Side chains from helices make connections with DNA bases in the groove H-bonds Zinc finger domains Beta sheet and alpha helix Use centrally coordinated zinc atoms Bound by 2 Cys from beta sheet and 2 His from alpha-helix Only large enough to bind a few DNA bases Often found in tandem repeats as part of a larger DNA binding region Rests in major groove of DNA  Amino acid side chains connect to bases in DNA Identity of side chain determines which bases are bound Assembling di;erent zinc fingers allows for greater specificity of protein RNA Splicing Eukaryotic genes are split Not all RNA that is transcribed is used Splicing Introns need to be removed Exons stay and code for the protein 5' cap is added to 5' end Why must introns be removed Often contain stop codons Proteins would be incomplete May shift translational reading frame of downstream exons Cells will stop growing and die Classification of introns Group 1 Group 2 Spliceosome Catalyse splicing Composition 150 proteins 5 RNAs Small nuclear (snRNA) U1, U2, U4, U5 & U6 100-300 nucleotides long Attach to proteins to form snRNPs Small nuclear ribonuclear proteins Pronounced snurps Named after snRNA they contain Carry out splicing How are introns recognised Splice site consensus sequences Most introns have the same general structure  Consensus sequences are recognised by snRNPs (snurps) How are separate ends of the intron brought together Spliceosome cycle Overview Mechanisms of RNA splicing Phosphodiester bond between 5’ exon and intron is broken Group 1 1. Intro, folds in such a way as to hold a free guanine nucleotide in ribose form 2. Guanine nucleotide OH group reacts with 5’ splice site 3. Guanine nucleotide attaches to 5’ end of intron 4. 3’ end of 5’ exon reacts with 3’ splice site Known as self splicing No other proteins are involved Group 2 May occur by self splicing or using spliceosome Self splicing Adenine residue is present in the sequence Opposed to using free adenine Intron forms a lariat (loop-like structure) by attaching to adenine in the sequence 3’ end of 5’exon attaches to 3’ splice site Spliceosome U1 binds to consensus sequence at 5’ splice site U2AF binds to 3’ splice site U2 auxiliary factor U2AF facilitates BBP binding to branch site BBP - Branch point binding protein Branch Point - Region of intron with adenine residue U2 displaces BBP at branch site Causes adenine at branch site to bulge U2AF is released and U2 recruits U4, U5, and U6 snRNPs U6 occupies same area as U1 As a result, U1 is released from 5’ splice site U6 attempts to interact with U2 As a result, U4 is released 5’ splice site breaks and a lariat is formed 3’ end of 5’ exon binds to 3’ exon