RNA Synthesis Lecture Notes PDF

BMN1001 Lecture 18: RNA synthesis- Transcription Dr. Dinesh Balachandra Lecturer (Non-Clinical) Biomedical Sciences NUMed Malaysia Email: [email protected]  What was covered in L14-17 The constituents of DNA (phosphate, deoxyribose sugar, base...

BMN1001 Lecture 18: RNA synthesis- Transcription Dr. Dinesh Balachandra Lecturer (Non-Clinical) Biomedical Sciences NUMed Malaysia Email: [email protected]  What was covered in L14-17 The constituents of DNA (phosphate, deoxyribose sugar, bases, nucleosides, nucleotides) Chargaff's rules and base pairing Fibre diffraction patterns of DNA helped to understand it’s structure The Watson-Crick double helical model of DNA DNA Sequencing Technologies 2 RNA Synthesis: Transcription DNA transcription mRNA protein Biological function 3 What you need to understand from this lecture (L18) Components of the system for RNA synthesis RNA polymerase & sigma factors promoters and consensus sequences Transcription initiation, elongation and termination 4 RNA structure 5  RNA Chemically, RNA is very similar to DNA BUT there are some key differences:  RNA is single-stranded (but can fold back on itself to form complex structures)  RNA uses the pentose sugar ribose instead of deoxyribose in the phosphodiester backbone  This renders RNA more unstable (see L15)  RNA uses the base Uracil (U) instead of Thymine (T). U is also complementary to A 6  The Components of Ribonucleotides 1) Heterocyclic base 2) Sugar 3) Phosphate _ O _ DNA: A, G, C, T and deoxyribose O P _ O RNA: A, G, C, U and ribose O 7 Polymeric structure of DNA and RNA The linkage that forms the polymer: 3’,5’-phosphodiester bonds 8 RNA forms long chains, like DNA uracil replaces thymine in base-pairing: U-A base pairs like T-A & Non- Watson-Crick base pairs can also form in RNA (Eg G/U (most common), G/T, C/U….. RNA forms secondary structures…. 9 RNA Structure 10  RNA secondary structures Stem-loop Bulge Single stranded but:  Folds to produce complex structures Tertiary interaction Tertiary - stem-loops Stem-loop interaction - bulges  can get non Watson-Crick base-pairs (variation on normal H-bonded structure)  “Tertiary” interactions 11 Measuring DNA and RNA Size Double-stranded DNA is measured in number of base pairs (bp): 1000bp= 1kbp 1000kb = 1Mbp RNA is measured in number of bases (b) 1000b = 1kb 1kb of mRNA codes for ~ 30kD protein 12  DNA vs RNA stability DNA and RNA stability refers to how well these molecules can withstand degradation and damage from various factors DNA is more stable than RNA mainly due to its double helix (double stranded) structure and enzymes are normally present in the nucleus to repair damage to DNA 13  DNA vs RNA stability There are no comparable enzymes in the nucleus or cytoplasm to repair damage to RNA and therefore RNA must be continuously replaced as they are broken down The presence of OH group in the carbon 2’ position makes RNA less stable compared to DNA making it more reactive and susceptible to hydrolysis 14  Classes of RNA produced in cells mRNA: messenger RNA, encodes proteins And many non-coding RNAs in prokaryotes and eukaryotes….. rRNA: ribosomal RNA, structural component of ribosome tRNA: transfer RNA, adaptor of info between mRNA and protein snRNA: small nuclear RNA, nuclear functions, including mRNA splicing snoRNA: small nucleolar RNA, process/modifies rRNA others: variety of processes (e.g. telomeres, gene inactivation, protein transport to ER) 15 What do you remember about transcription? DNA transcription mRNA protein Biological function 16 The presence of an OH group on Carbon 2 of the pentose sugar ribose causes…. A. DNA to be stable B. DNA to be unstable C. RNA to be stable D. RNA to be unstable E. there to be more DNA in the cell than RNA 17  Which of these do you think are more abundant in a cell? A. dNTPs (deoxyribonucleic acids) B. NTPs (ribonucleic acids) 18  Which has the highest error rate? A. DNA polymerases B. RNA polymerases 19  Which of the following statements are true about RNA polymerase in E. coli? A. RNA polymerase consists of one subunit B. RNA polymerase contains sites that directly recognize the promoter sequence C. Only one RNA transcript can be made from a gene at a time D. RNA polymerase has to associate with a Sigma factor in order to recognize a specific promoter sequence 20  In an E. coli genome (or any other genome), the promoter of a particular gene or operon is located… A. 3’ of a gene or operon to be transcribed, on the same DNA strand B. 5’ of the gene or operon to be transcribed, on the same DNA strand C. 3’ of the gene or operon to be transcribed, on the opposite DNA strand D. 5’ of the gene or operon to be transcribed, on the opposite DNA strand 21 RNA polymerases 22 Ingredients for Transcription rNTPs DNA template Enzymes RNA Polymerase 23  Transcription makes RNA from DNA RNA is synthesised by RNA polymerase Newly transcribed RNA molecules are released from DNA RNA synthesis occurs 5’ to 3’ doesn’t need primers RNA polymerase has an error rate of ~ 1 x 10-4 Lots of RNA molecules are made from 1 DNA molecule 24 Transcription makes RNA from DNA 25  Transcription makes RNA from DNA highly selective  RNA is synthesised from one DNA strand (TEMPLATE STRAND) =ANTI-SENSE STRAND  RNA is synthesised complementary and anti-parallel to template (anti-sense) strand Sense strand = same sequence as mRNA 26  RNA polymerases Eukaryotes have three different RNA polymerases which transcribe different sets of genes RNA pol I - ribosomal RNA (5.8, 18 and 28S rRNA) RNA pol II - all protein coding genes RNA pol III - tRNA (+ other non-coding RNAs) Prokaryotes have one RNA polymerase 27 Transcription in E.coli 28  Why Study Gene Expression in E. coli? Bacteria are key human pathogens Knowledge of the process of gene expression in E. coli provides insight into this process in pathogens The bacterial gene expression machinery is the target for some antibiotics rifampicin CEM-101 - translation streptolydigin transcription lipiarmycin E. coli is an important host for the production of “recombinant” (foreign) proteins e.g. Humulin: human insulin produced in E. coli Understanding control of gene expression and its control in E. coli provides a framework for understanding it in more complex organisms 29  Transcriptional regulation What can be transcribed? A single gene An Operon- one or more genes transcribed into a single RNA and under the control of a single regulatory site A Regulon- a series of operons controlled as a unit A Stimulon - a collection of genes (in operons and regulons) under regulation by the same stimulus  NB. These were thought to occur only in prokaryotes, but in 1990s a few operons were discovered in some eukaryotes- e.g C. elegans, Drosophila melanogaster, some rRNAs – bacteria have a lot to teach us! 30  Transcription- some definitions Transcription (DNA to RNA) is carried out by the enzyme RNA polymerase – Uses DNA as template (one strand of each gene), makes RNA from precursors ATP, GTP, CTP, and UTP, chain growth is 5′ to 3′ like DNA replication – Significant differences between RNA polymerases in Bacteria, Archaea, Eukarya- bacterial RNA polymerase has simplest structure Promoter = transcription initiation site on the DNA molecule, where RNA polymerase initially binds Operator = a segment of DNA to which a transcription factor protein binds between the promoter and the genes of the operon Transcription terminators = specific sites - transcription stops Transcriptional activators = proteins that facilitate the binding of RNA polymerase or increase the number of transcripts Transcriptional repressors = proteins that repress binding of RNA polymerase 31 Steps in RNA synthesis: Initiation Elongation Termination Lots of transcription events can be going on at the same time! 32  Transcription in E. coli Transcription START 5’ 3’ 3’ 5’ DNA promoter transcribed region terminator (1) Promoter (2) Transcribed region -mRNA can be POLYCISTRONIC (3) Terminator 33 Transcription promoters- reminder PROMOTER = Region of DNA upstream (5’) of a gene that contains specific nucleotide sequences with which transcription factors can associate. These factors then recruit RNA polymerase Can identify conserved regions- consensus sequences 34 How are consensus sequences determined ? Sequence comparison: -35 region promoter 1 T T G T C A promoter 2 T T G A C T promoter 3 T A G G C A promoter 4 T G G A A A promoter 5 G T T A C A promoter 6 T T G A T A promoter 7 T T G A C C CONSENSUS T T G A C A 35 Identifying consensus sequences in bacterial promoters 36 E. coli RNA polymerase RNA polymerase Mg2+ dependent and multisubunit Core RNA polymerase is composed of: 2xa 1xb 1 x b’ 1xw Core + s factor = Holoenzyme 37  Transcription- RNA polymerase RNA polymerase is a multisubunit - core enzyme of 5 different subunits- β, β’, α (x2) and ω (omega), associated with the sigma (σ) factor to form RNA polymerase holoenzyme. Sigma factor = subunit responsible for promoter binding binds to two shorter sequences within the promoter which are highly conserved: -35 and -10 sequences (-10 = Pribnow box) interaction of RNA polymerase with the promoter 38  Sigma factors Different sigma (s) factors allow expression of different genes/ groups of genes E.g. the major sigma factor in E.coli (best studied) is s70 (ie 70kDa sigma factor) for normal gene expression- but it uses other types as well Transcriptional activators that interact with RNA polymerase containing s70 bind close to RNA polymerase and stabilise binding to the promoter 39  The E. coli s70 Promoter -35 sequence -10 sequence start-site (+1) TTGACA TATAAT TG/AT 16-19bp 5-8bp consensus sequences 40  Domains of σ 70 Interactions with core RNA polymerase N- 1.1 1.2 2.2 2.3 2.4 4.1 4.4 -C Prevents binding to Mediate binding to the promoters in the absence Mediate binding to -35 element of RNA polymerase the -10 element The function of various domains in σ70 have been determined These mediate the binding of RNA polymerase to different classes of promoter Although σ70 is the main sigma factor other “alternative” sigma factors exist (e.g. σ32 and σ54) 41  Alternative sigma factors Most genes in E. coli require the standard sigma factor s70 However, E. coli also has several other sigma factors that recognise different consensus sequences Alternative sigma factors are specific for a group of genes required under special circumstances E.g. s38 recognises a consesnsus sequence found in genes expressed during stationary phase Activity of sigma factors can be temporarily blocked by other proteins- anti-sigma factors- or factors controlling rate of synthesis/ degradation of the sigma factor 42  Sigma factors in E. coli Seven different sigma factors in E. coli recognising different consensus sequences Most of these have counterparts in other bacteria 43  INITIATION- KEY POINTS During transcription one strand of DNA is copied into RNA by RNA polymerase The promoter is the binding site for RNA polymerase The promoter sequences specify the efficiency of transcription RNA polymerase binding to the promoter requires the sigma-factor The sigma-factor reduces affinity of RNA polymerase for non-specific DNA 44 Formation of the Transcription Complex- Initiation and Elongation Closed RNA polymerase Complex 5’ s sense strand 3’ 3’ 5’ -35 -10 +1 antisense (template) strand Open INITIATION Complex s -35 -10 ELONGATION s 3’ -35 -10 +1 45 5’ mRNA Elongation: 5’ 3’ Chain Growth  Elongation Transcription is rapid (20-50 nt per sec at 37◦C) in E. coli RNA polymerase has no proofreading function (?) Error rate is approx 1 in 10 000 47 Structure of the Elongation Complex: 48  Termination RNA polymerase stops transcribing at terminator sequences Two types of termination: (1) Factor-independent termination: Formation of a stem-loop structure by complementary base-pairing (2) Rho Dependent; depends on the Rho protein factor 49  Factor Independent Termination Common features of factor independent terminators (1) Series of 4 to 10 consecutive A-T base pairs (2) A G+C rich region with a palindromic sequence that immediately precedes the series of A-T base pairs N N N DNA template N N 5’ NNAAGCGCCGNNNNNCGGCGCTTTTTTNNN 3’ G-C 3’ NNTTCGCGGCNNNNNGCCGCGAAAAAANNN 5’ C-G C-G 5’ NNAAGCGCCGNNNNNCGGCGCUUUUUUNNN 3’ G-C C-G RNA transcript G-C A-U A-U ---NNNN UUUU- 3’OH 50 Factor Independent Termination 51 Factor Independent Termination G C C CG G C U U U U U C G G GC N N N N N 52 Factor Independent Termination C G C U U U U U C G CG G CG GC N N N N N 53 Factor Independent Termination U U U U U GC CG GC CG CG GC N N N N N 54  Rho (r) dependent Termination The Rho factor is composed of six identical subunits Rho is a helicase that unwinds RNA-DNA and RNA-RNA duplexes Powered by the hydrolysis of nucleoside triphosphates 55  Rho (r factor) dependent Termination Rho loads onto C-rich sequences RUT (Rho utilisation site) RNA polymerase pauses at ter site Rho unwinds the RNA:DNA hybrid RNA pol, mRNA and Rho are released 56  What we covered today (L18) RNA structure Components of the system for RNA synthesis – RNA polymerase + sigma factors – promoters and consensus sequences – Transcription initiation, elongation and termination Next lecture L19 – How transcription (and so gene expression) is controlled 57  Points for Revision - BMN1001 L18 Do you know what a promoter is and where its key sequence elements are located? Do you know what a consensus sequence is? How does the promoter sequence influence its strength Do you know the subunit composition of core RNA polymerase? Do you know what subunit allows RNA polymerase to recognise promoter elements? What is this form of RNA polymerase called?? Can you describe the process of transcription initiation in terms of closed and open complexes? When is the sigma-factor is released from RNA polymerase? Can you describe the process of transcriptional elongation? Can you describe how self complementary sequences in the mRNA form a hairpin & aid termination? Do you know what the Rho factor is and how it functions? 58  Further reading (L13-L21) Molecular Biology of the Cell 6th Edition (Garland Science): Chapter 2: Cell Chemistry and Biosynthesis (Panels on chemical bonding etc.) Chapter 4: DNA, Chromosomes + Genomes Hard copies in the LRC or use the direct link: (need uni login) https://libsearch.ncl.ac.uk/primo-explore/fulldisplay?docid=NCL_ALMA51148399790002411 &context=L&vid=NEWUI&lang=en_US&search_scope=NotPC&adaptor=Local%20Search %20Engine&isFrbr=true&tab=default_tab&query=any,contains,Molecular%20Biology%20of %20the%20Cell&sortby=date&facet=frbrgroupid,include,3074405341&offset=0 Biochemistry, 7th Edition Berg, Tymoczko and Stryer W. H. Freeman, 2011 Chapter 29: RNA synthesis Link: https://www.academia.edu/38170707/Biochemistry_8th_ed_pdf 59  Any Questions? Dr. Dinesh Balachandra Room 3.50, Bell’s Court Tel: + 607 555 3800 extn. 3825 Email questions welcome! Email: [email protected]