Gene Expression Student Notes PDF

School of Biosciences Gene expression Dr Andy Bates BIOS101 School of Biosciences Genes produce proteins The idea pre-dates the DNA structure Beadle and Tatum (1941) – Mutations in Neurospora crassa can be localise...

School of Biosciences Gene expression Dr Andy Bates BIOS101 School of Biosciences Genes produce proteins The idea pre-dates the DNA structure Beadle and Tatum (1941) – Mutations in Neurospora crassa can be localised to a particular point on a chromosome – They correleate with defects in particular enzymes in biosynthetic pathways. School of Biosciences Central dogma - Crick tRNA rRNA snRNA School of Biosciences Codons are 3 nucleotides 42 = 16 43 = 64 20 amino acids School of Biosciences Har Gobind Khorana – 1968 Nobel Prize School of Biosciences Deducing the code Synthesise 5´-UCUCUCUCU… This results in SerLeuSerLeu… This tells you that UCA and CUC must code for serine and leucine… School of Biosciences Genetic code Also no punctuation i.e. AAABBBCCC not AAAxBBBxCCCx Non-overlapping i.e. AAABBBCCC not AAA ABB BCC School of Biosciences Reading frames --CAGAAUAUCAUCUAUUCUACA-- 1. asn ile ile tyr ser thr 2. ile ser ser ile leu xxx 3. tyr his leu phe tyr xxx School of Biosciences Genetic code School of Biosciences Genetic code Degenerate Universal Optimised to minimise effect of mutations Third position often does not matter First position changes result in similar chemistry School of Biosciences Bacterial DNA School of Biosciences Bacterial DNA Circular DNA Packaged by supercoiling, and wrapping round some proteins Fairly open structure Default position is that genes are on - transcribed School of Biosciences RNA polymerase Core polymerase a2bb'ω + Sigma proteins (σ) σ Holoenzyme a2bb'ωσ School of Biosciences Genes and promoters Coding strand TATAAT Sense strand Top strand 5'--TTGACA----TATAAT----AGGATT-------- 3'--AACTGT----ATATTA----TCCTAA-------- Template strand Antisense strand -35 -10 +1 Bottom strand 5' AGGAUU-------- PROMOTER RNA RNA polymerase recognises promoter Unwinds from -10 position Starts RNA synthesis at +1 School of Biosciences RNA polymerase   Holoenzyme Core  enzyme ‫׳‬  Prokaryotic RNA polymerase School of Biosciences Initiation of transcription Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.  binds to DNA RNA polymerase bound TATAAT TATAAT–Promoter (–10 sequence) to unwound DNA Transcription bubble 5′ Downstream Template 5′ 3′ 3′ strand Coding strand Start site (+1)  dissociates ATP TTGACA TTGACA–Promoter Helix opens at (–35 sequence) –10 sequence Start site RNA synthesis begins Upstream 5′ 3′ School of Biosciences RNA polymerase DNA Start site Codingstrand Codingstrand Unwinding Unwinding Rewinding Rewinding 3' 5′ 3' 5′ Downstream 3' Upstream Template Template strand strand mRNA Transcription bubble 5′ School of Biosciences Specific DNA binding The Helix-Turn-Helix Motif α Helix Turn (Recognition helix) α Helix Turn α Helix 3.4 nm 90° a. School of Biosciences Control of gene expression E.coli lac operon (Jacob and Monod) Expresses the genes to use lactose as a carbon source CAP-binding site Gene for Operator repressor Gene for protein Promoter for permease Promoter lac operon for I gene Genefor ß-galactosidase Gene for transacetylase CAP Plac O PI I Z Y A Regulatory region Coding region lac Control system School of Biosciences Lactose Absent lac Operon Is Repressed lac repressor polypeptide lac Repressor mRNA CAP–binding site (No lactose present) cAMP lac CAP repressor lac repressor gene Promoter for No transcription lac operon Enzymes to metabolize lactose not produced DNA Z Y A RNA polymerase is blocked by the lac repressor Operator Lactose Present lac Operon Is Induced Allolactose (inducer) ß-Galactosidase mRNA (lactose present) Translation Transacetylase Permease lac Repressor cannot bind to DNA Enzymes to metabolize mRNA lactose produced Z Y A RNA polymerase is not blocked and transcription can occur School of Biosciences School of Biosciences tRNA adapters 2D “Cloverleaf” Model 3D Ribbon-like Model Acceptor end Acceptor end 3′ 5′ Anticodon loop Anticodon loop Amino acyl tRNA synthestases School of Biosciences The ribosome Large subunit 3′ 90 ° Small Large subunit subunit Small Large subunit subunit 0° Small mRNA subunit 5′ School of Biosciences Chemistry of translation Amino end NH3+ Amino (N terminus) group NH3+ Amino acid 1 Amino O C acid 1 Peptide NH3+ Polypeptide N Amino NH3+ bond chain acid 2 Amino Amino Amino Amino acid 2 acid 2 acid 1 acid 3 C O C O O C 3′ Amino O “Empty” O acid 4 Peptide OH O tRNA bond Amino formation acid 5 Amino acid 6 5′ Amino acid 7 COO– A site Carboxyl end P site (C terminus) School of Biosciences Eukaryotic gene expression Transcription and translation separate – Nucleus and cytoplasm Message is processed – Capping, poly-A tail, splicing Chromatin structure – 1st level of control School of Biosciences Chromatin organisation School of Biosciences Chromatin DNA wrapped around histone octamers to make nucleosomes Nucleosomes wrapped into tight 30 nm fibre Most DNA is wrapped in fibres most of the time School of Biosciences DNA accessibility DNA methylation 5-methyl C in 5´-CG-3´ – Marker of inactive DNA H CH 3 H N O H N N G N H N C H Phosphate N N N H O H School of Biosciences DNA accessibility Histone modification – Methylation promotes tighter packaging – Acetylation promotes opening – gene expression – Chromatin remodelling ATP-dependent Nucleosome sliding Displacement School of Biosciences Eukaryotic transcription Requires stimulation by transcription factors and activators School of Biosciences Eukaryotic message processing 5´-cap and poly-A tail 5′cap HO OH P 7-methylguanine (7-Me G) P P CH2 a il + y -At 3′ ol N+ 3′ p AA A AA CH3 Methyl group AA P mRNA P P G 5′ CH3 School of Biosciences Splicing snRNPs In nucleus School of Biosciences Alternative splicing Genes can produce multiple related proteins 90 % of human genes may be alternatively spliced. 1 1 2 2 3 3 4 4 5 5 6 5´cap 3´ Poly- A tail Primary RNA transcript Introns are spliced Exons Introns Thyroid splicing pattern Hypothalamus splicing pattern 1 2 3 4 1 2 3 5 6 3´ Poly- A tail 5´cap 3´ Poly- A tail Mature mRNA Mature mRNA Calcitonin CGRP School of Biosciences Summary The genetic code is how DNA specifies proteins (through the intermediary mRNA) The code is universal and optimised Prokaryotic genes are controlled by repression of transcription. Eukaryotic chromatin represses most transcription Transcription is controlled by multiple factors Alternative splicing can derive multiple proteins from one gene.