BIOL2010 Transcription Lecture 1 v4 PDF

BIOL2010 – Transcription Lecture 1 – Transcription overview Dr Declan A. Doyle E-mail: [email protected]  Recommended Books  Molecular Biology of the Gene – Watson  Genomes - Brown  Molecular Biology of the cell - Alberts Lots more in the BIOL2...

BIOL2010 – Transcription Lecture 1 – Transcription overview Dr Declan A. Doyle E-mail: [email protected]  Recommended Books  Molecular Biology of the Gene – Watson  Genomes - Brown  Molecular Biology of the cell - Alberts Lots more in the BIOL2010 Bb Reading List  Transcription Lecture 1/2: Overview of transcription Transcriptional control in bacteria Post-transcriptional control overview Lecture 3/4: Transcription in eukaryotes Promoter structure Transcription factors Lecture 5/6: Eukaryotic post-transcriptional control 3’ processing, RNA splicing 5’ Cap, transport to cytoplasm  Lecture 1  Overview of transcription  Transcriptional control in bacteria  Promoters  RNA Polymerase How does DNA encode for proteins? Replication DNA How does DNA encode for proteins? Replication DNA Transcription RNA How does DNA encode for proteins? Replication DNA Transcription RNA Translation Protein  How does DNA encode for proteins? EUKARYOTIC DNA contains the code for proteins DNA is in the nucleus Proteins are made in the cytoplasm How does the message get from nucleus to cytoplasm? mRNA  How does DNA encode for proteins? PROKARYOTIC Replication DNA Transcription RNA Replication + Translation transcription + translation all happens in Protein the cytoplasm. Why is the intermediate mRNA required? 1. Better regulation of the Replication protein production process. 2. Fidelity of DNA replication is DNA very important. Having an intermediate step prevents interference and potential additional mistakes. 3. Having an intermediate step allows for mistakes in protein production. Protein 4. Proteins can still be made when DNA is being replicated.  What is RNA? DNA Transcription RNA What is RNA?  RNA is a linear polymer like DNA  Residues linked by phosphodiester bonds  Contains ribose not deoxyribose  RNA is single stranded What is RNA?  Contains: A (adenine) G (guanine) C (cytosine)  but not T (thymine); uses U (uracil) instead  U base-pairs with A (just like A:T base pair)  Why does biology use uracil instead of thymine in RNA? What is RNA?  Although RNA is single stranded, it can fold into specific structures  This involves base- paring and covalent bonds  This allows some RNA molecules to have structural and catalytic functions  What types of RNA are there?  rRNA: Ribosomal RNA, form the basic structure of the ribosome and catalyse protein synthesis  tRNA: Transfer RNAs, central to protein synthesis as adaptors between mRNA and amino acids  Non-coding RNA, e.g. microRNAs, long non-coding RNAs  mRNA: Messenger RNA, codes for proteins (3-5%) The Nobel Assembly at Karolinska Institutet Awarded the 2024 Nobel Prize in Physiology or Medicine jointly to: Victor Ambros and Gary Ruvkun for the discovery of microRNA and its role in post-transcriptional gene regulation  How does DNA encode for proteins? EUKARYOTIC MicroRNA Long non-coding RNA (>200 nt, not transcribed. Involved in regulation through binding RNA, proteins and DNA thus influencing important interactions)  How does DNA encode for proteins?  Regulation of transcription is the most common form of control of protein production MicroRNA  How is RNA produced? DNA Transcription RNA The Nobel Prize in Physiology or Medicine 1959 Severo Ochoa and Arthur Kornberg "for their discovery of the mechanisms in the biological synthesis of ribonucleic acid and  How is RNA produced? A gene: entire nucleotide sequence required to direct protein synthesis DNA 5’ Promoter region Coding region 3’ Control region 5’ RNA Polymerase 3’ 3’ 5’ ATG TAG TAA TGA Transcription initiates RNA  The structure of RNA polymerase Crab claw Thermus aquaticus Yeast PROKARYOTIC EUKARYOTIC RNA Polymerase  Separates the 2 strands of DNA  Uses one of the DNA strands as a template for RNA synthesis  Does not require a primer  It is very accurate 1/10,000 bases  RNA Polymerase  Moves along the gene in a 5’ to 3’ direction  Synthesises a complementary RNA copy of the DNA template strand  Many RNA copies are made at the same time Electron-micrograph of transcriptions of 2 genes  Multiple RNA polymerases per gene  Multiple transcripts per gene  as soon as the first RNA polymerase starts to move down the gene, the next polymerase can bind and initiate RNA transcription  Mechanism of Transcription RNA polymerase Coding strand CCGTA 5’ 3’ 3’ 5’ GGCAT Template strand  Mechanism of Transcription RNA polymerase Coding strand CCGTA 5’ 3’ 3’ 5’ GGCAT Template strand Unwinding  Mechanism of Transcription RNA polymerase Coding strand CCGTA 5’ 3’ 3’ 5’ GGCAT Template strand Unwinding Movement of polymerase  Mechanism of Transcription RNA polymerase Coding strand CCGTA 5’ 3’ 3’ 5’ GGCAT Template strand Unwinding Movement of polymerase  Mechanism of Transcription RNA polymerase Coding strand CCGTA 5’ 3’ 3’ 5’ GGCAT Template strand C G C Movement of A polymerase U  Mechanism of Transcription RNA polymerase Coding strand Rewinding CCGTA 5’ 3’ 3’ CCGUA 5’ GGCAT Template strand Movement of polymerase  Mechanism of Transcription RNA polymerase Coding strand Rewinding CCGTA 5’ 3’ 3’ CCGUA 5’ GGCAT Template strand Movement of polymerase  Length of the bubble is 12-14bp  Reaction rate approx 40 bases per sec  Mechanism of Transcription RNA polymerase Coding strand CCGTA 5’ 3’ 3’ 5’ 3’ Template strand RNA-DNA hybrid helix 5’ppp Nascent RNA Elongation site Mechanism of Transcription Transcription proceeds in 3 main steps 1. Initiation - Template recognition Binding - Polymerase locates closed promoter complex Separatin g- open complex Polymerase unwinds the DNA Transcription begins Transcription proceeds in 3 main steps 2. Elongation Polymerase places RNA in exit hole Once RNA is 10 or more base pairs long Polymerase conformation change - tightens grip Transcription proceeds in 3 main steps 3. Termination Termination sequence Polymerase separates RNA released  Transcription is similar in: Prokaryotes Differences: Initiation Promotor RNA Polymerase Eukaryotes  Similar Transcriptional Control Control of transcription is the major way protein expression is controlled More protein required mRNA synthesis Protein synthesis Less protein required mRNA synthesis Protein synthesis  This ability to control the level of transcription is key More protein required mRNA synthesis Protein synthesis  The importance of control E. coli pET protein over-expression system 1. Clone your gene into pET vector; 2. Transform E. coli BL21(DE3); 3. Grow cells (more cells = more protein); 4. Add inducer (IPTG) The importance of control Protein over-expression in Escherichia coli triggers adaptation analogous to antimicrobial resistance Protein over-expression in Escherichia coli triggers adaptation analogous to antimicrobial resistance >99.9 % of all cells die = Protein over-expression in Escherichia coli triggers adaptation analogous to antimicrobial resistance  Summary  The transcription processes for both bacteria and eukaryotes go through the same 3 major steps: 1. Initiation 2. Elongation 3. Termination  The most important regulation step in both cases is the number of mRNA transcripts generated.  Generally speaking, more mRNA = more protein  Regulation is important as both under-production and over-production of mRNA can have serious effects.