BB1725 Lecture 6 RNA and transcription (4) PDF

BB1725 Biology of the Cell Lecture 6: RNA and Transcription Dr Joseph Hetmanski [email protected] Today’s Lecture In today’s lecture: Differences between RNA and DNA Transcription - how mRNA is made Types of RNA By the end of the lecture,...

BB1725 Biology of the Cell Lecture 6: RNA and Transcription Dr Joseph Hetmanski [email protected] Today’s Lecture In today’s lecture: Differences between RNA and DNA Transcription - how mRNA is made Types of RNA By the end of the lecture, Figure 6–9 DNA is transcribed by the enzyme RNA you should be able to explain polymerase Molecular Biology of the Cell - Seventh Edition - Copyright © how the cell makes mRNA 2022 W.W. Norton & Company, Inc. from DNA Brunel University London – BB1725 Biology of the Cell – Lecture 6 – 2024/25 2 Transcription and Translation Genetic information is expressed in the form of proteins by a two stage process: Transcription Generates a single-stranded RNA identical in sequence with one of the strands of the DNA duplex Translation Converts the RNA sequence into the Figure 6–1 Genetic information directs the synthesis of proteins amino acid sequence comprising a Molecular Biology of the Cell - Seventh Edition - protein Copyright © 2022 W.W. Norton & Company, Inc. Brunel University London – BB1725 Biology of the Cell – Lecture 6 – 2024/25 3 Dictionary definitions Transcription: a written or printed version of something. Translation: the process of translating words or text from one language into another. Brunel University London – BB1725 Biology of the Cell – Lecture 6 – 2024/25 How does RNA differ from DNA? Figure 6–5A The chemical structure of RNA Molecular Biology of the Cell - Seventh Edition - Copyright © 2022 W.W. Norton & Company, Inc. Brunel University London – BB1725 Biology of the Cell – Lecture 6 – 2024/25 5 DNA vs RNA Brunel University London – BB1725 Biology of the Cell – Lecture 6 – 2024/25 How does RNA differ from DNA? Figure 6–5B The chemical structure of RNA Molecular Biology of the Cell - Seventh Edition - Copyright © 2022 W.W. Norton & Company, Inc. Brunel University London – BB1725 Biology of the Cell – Lecture 6 – 2024/25 7 In RNA, U replaces T In RNA, uracil (U) replaces thymidine (T) Uracil (U) will bind with adenine (A) Uracil is less stable than Thymine, which is useful for shorter lived Figure 6–6 Uracil base pairs with adenine RNA Molecular Biology of the Cell - Seventh Edition - Copyright © 2022 W.W. Norton & Company, Inc. Brunel University London – BB1725 Biology of the Cell – Lecture 6 – 2024/25 8 Transcription Involves formation of complementary bp of RNA using antisense DNA template Carried out by RNA polymerase enzymes Figure 6–8 DNA transcription produces a single-strand RNA molecule that is complementary to one strand of the DNA double helix Molecular Biology of the Cell - Seventh Edition - Copyright © 2022 W.W. Norton & Company, Inc. Brunel University London – BB1725 Biology of the Cell – Lecture 6 – 2024/25 9 Products of gene expression Segments of DNA (“transcription units” or “genes”) are transcribed as discrete units as and when required The products of some genes are messenger RNA molecules (mRNA) that are subsequently translated into proteins – these are ‘coding genes’ For other non-coding genes (e.g. tRNA or rRNA or miRNA genes), the RNA itself is the functional end product Brunel University London – BB1725 Biology of the Cell – Lecture 6 – 2024/25 1 0 RNA polymerase RNA polymerases are multi unit enzymes which synthesize new RNA from DNA templates Eukaryotes have three RNA polymerases: RNA polymerase I (ribosomal) rRNA genes (5.8S, 18S and 28S) RNA polymerase II All protein-coding genes (messenger mRNA) Small non-coding RNAs – e.g. some microRNA (miRNA) RNA polymerase III (Transfer) tRNA, 5S rRNA and some miRNA S = Svedberg unit – the rate of sedimentation (i.e. = size Brunel University London – BB1725 Biology of the Cell – Lecture 6 – 2024/25 1 1 RNA polymerase structure Figure 6–9 DNA is transcribed by the enzyme RNA polymerase Molecular Biology of the Cell - Seventh Edition - Copyright © 2022 W.W. Norton & Company, Inc. Figure 6–11B The transcription cycle of bacterial RNA polymerase Molecular Biology of the Cell - Seventh Edition - Copyright © 2022 W.W. Norton & Company, Inc. Brunel University London – BB1725 Biology of the Cell – Lecture 6 – 2024/25 12 Generalised eukaryotic gene structure Transcription start site Poly A tail signal Transcription end DNA Exon Intron Promoter Terminator (Gene Control) (Gene Control) Exons - are expressed and make proteins Introns - are not expressed/translated Brunel University London – BB1725 Biology of the Cell – Lecture 6 – 2024/25 13 Transcription explanation https://www.youtube.co m/watch?v=XzVXhemt wmA Brunel University London – BB1725 Biology of the Cell – Lecture 6 – 2024/25 Promoters contain control regions Certain regions contain sequences which bind to transcription machinery: Initiator element around transcription start site (C at -1 and A at +1) TATA box (TATAAA) around 25-35 bp upstream of transcription start site Transcription Factor IID (TFIID) – which contains the TATA binding protein (TBP) - binds here to initiate transcription Followed by other basal transcription factors and RNA Polymerase II Upstream regulatory elements (100-200 bp upstream) GC box (GGGCGG) binds SP1 transcription factor CAAT box (CCAAT) binds many transcription factors Figure 6–15 Initiation of transcription of a eukaryotic gene by RNA polymerase II Molecular Biology of the Cell - Seventh Edition - Copyright © 2022 W.W. Norton & Company, Inc. Brunel University London – BB1725 Biology of the Cell – Lecture 6 – 2024/25 15  TABLE 6–3 The General Transcription Factors Needed for Transcription Initiation by Eukaryotic RNA Polymerase II Molecular Biology of the Cell - Seventh Edition - Copyright © 2022 W.W. Norton & Company, Inc. Brunel University London – BB1725 Biology of the Cell – Lecture 6 – 2024/25 16 Other control elements Enhancers Activate transcription May be quite distant from the promoter (several kb), upstream or downstream Enhancers upregulate transcription Silencers Repress transcription Equivalent regulatory elements that inhibit transcription Brunel University London – BB1725 Biology of the Cell – Lecture 6 – 2024/25 17 mRNA processing First step - make the RNA But… How do we go from the primary transcript (pre- mRNA) to the mature mRNA molecule? Figure 6–21A Comparison of the steps leading from gene to protein in eukaryotes and bacteria Molecular Biology of the Cell - Seventh Edition - Copyright © 2022 W.W. Norton & Company, Inc. Brunel University London – BB1725 Biology of the Cell – Lecture 6 – 2024/25 18 Step 1: Initiation of Transcription Activators may be 1000s of bases upstream More than one activator needed Mediator helps assembles the required proteins Figure 6–18 Transcription initiation by RNA polymerase II in a eukaryotic cell Molecular Biology of the Cell - Seventh Edition - Copyright © 2022 W.W. Norton & Company, Inc. Brunel University London – BB1725 Biology of the Cell – Lecture 6 – 2024/25 19 Step 2: Make the pre-mRNA Translates DNA to pre-mRNA 1.3 and 4.3 kb min−1 Carries the pre-mRNA processing proteins on its ‘tail’ ‘Tail’ is phosphorylated to attract the proteins required Pre-mRNA processing proteins Figure 6–23 Eukaryotic RNA are transferred to the pre- polymerase II as an RNA synthesis and processing mRNA when needed machine Molecular Biology of the Cell - Seventh Edition - Copyright © 2022 W.W. Norton & Company, Inc. Brunel University London – BB1725 Biology of the Cell – Lecture 6 – 2024/25 20 Step 3: Capping As pre-mRNA emerges from the RNA polymerase it is capped 25 nucleotides appear Phosphatase removes 5’ phosphate Guanyl transferase adds GMP Methyl transferase adds methyl group to the guanosine 5’ cap helps ‘signal’ that this is Figure 6–24 The reactions that mRNA - no cap = rapid cap the 5′ end of each RNA molecule synthesized by RNA polymerase II degradation Molecular Biology of the Cell - Seventh Edition - Copyright © 2022 W.W. Norton & Company, Inc. Brunel University London – BB1725 Biology of the Cell – Lecture 6 – 2024/25 21 Step 4: RNA Splicing A specific adenine in the intron attacks the upstream splice site and cuts the sugar-phosphate backbone, attaches and forms a loop The free 3’ end of the exon reacts with the start of the next exon, therefore removing the intron Without splicing, the wrong Figure 6–26A The pre-mRNA splicing reaction protein may be formed later Molecular Biology of the Cell - Seventh Edition - Copyright © 2022 W.W. Norton & Company, Inc. Brunel University London – BB1725 Biology of the Cell – Lecture 6 – 2024/25 22 Spliceosome The Spliceosome carries out the splicing The Spliceosome consists of: Five additional RNA molecules - U1, U2, U4, U5 and U6 Several hundred proteins RNA molecules - U1, U2, U4, U5 and U6 Small nuclear RNA (snRNA) - ~200 nucleotides Perform key steps Recognise splicing site Forms complexes with the proteins Spliceosome uses ATP Figure 6–29 The pre-mRNA splicing mechanism Molecular Biology of the Cell - Seventh Edition - Copyright © 2022 W.W. Norton & Company, Inc. Brunel University London – BB1725 Biology of the Cell – Lecture 6 – 2024/25 23 Step 5: Polyadenylation RNA sequence at 3’ end recognised by RNA binding and processing proteins CPSF and CstF bind - recruit other proteins Pre-mRNA cleaved from the polymerase Poly-A polymerase adds ~200 A Required for stability of mature mRNA, export from the nucleus and Figure 6–37 Some of the major steps in generating the 3′ end of a eukaryotic mRNA subsequent transport Molecular Biology of the Cell - Seventh Edition - Copyright © 2022 W.W. Norton & Company, Inc. Brunel University London – BB1725 Biology of the Cell – Lecture 6 – 2024/25 24 Summary Transcription produces RNA from DNA using RNA polymerases Transcription initiation Pre mRNA production (from DNA) 5’ capping to prevent degradation Splicing out of introns to ensure correct proteins made 3’ polyadenylation to stabalise and facilitate export/transport A Brunel University London – BB1725 Biology of the Cell – Lecture 6 – 2024/25 25 Types of RNA rRNA: ribosomal RNA – makes up the ribosome (alongside many proteins) the machinery for making proteins mRNA: messenger RNA – the intermediary code from DNA to protein tRNA: transfer RNA – adapters, carries the amino acids to the ribosomes Others include microRNA All University Brunel involved in forming London – BB1725 Biology of the Cell proteins – Lecture 6 – 2024/25 transfer RNA - tRNA Figure 6–54 A tRNA molecule Molecular Biology of the Cell - Seventh Edition - Copyright © 2022 W.W. Norton & Company, Inc. Brunel University London – BB1725 Biology of the Cell – Lecture 6 – 2024/25 27 mRNA is decoded on Ribosomes Translation is performed on the ribosome, a complex protein synthesis machine made from more than 80 ribosomal proteins and several ribosomal Figure 6–64 Ribosomes in the cytoplasm of a eukaryotic cell Molecular Biology of the Cell - Seventh Edition - Copyright © 2022 W.W. Norton & Company, RNAs (rRNAs) Inc. Brunel University London – BB1725 Biology of the Cell – Lecture 6 – 2024/25 28 Ribosomes Ribosomes are composed of one large and one small subunit Made of ribosomal proteins and rRNA How were the ribosomal proteins made in the first place? Figure 6–65 A comparison of bacterial and eukaryotic ribosomes Molecular Biology of the Cell - Seventh Edition - Copyright © 2022 W.W. Norton & Company, Inc. Brunel University London – BB1725 Biology of the Cell – Lecture 6 – 2024/25 29 Ribosomes 4 binding sites: 1.one for the mRNA Three for tRNA : 2.A (aminoacyl)-site 3.P (peptidyl)-site 4.E (exit)-site Figure 6–66D The RNA-binding sites in the ribosome Molecular Biology of the Cell - Seventh Edition - Copyright © 2022 W.W. Norton & Company, Inc. Brunel University London – BB1725 Biology of the Cell – Lecture 6 – 2024/25 30 References Explore the material for this week on Brightspace Molecular Biology of the Cell by Alberts et al. 7th Ed. - chapter 6 Brunel University London – BB1725 Biology of the Cell – Lecture 6 – 2024/25 31 References and Videos Introns: https://www.ncbi.nlm.nih.gov/pmc/articles/P MC4742320/ Transcriptional initiation by RNA Pol II: https://www.youtube.com/watch?v=IdH6Zb hP7us Brunel University London – BB1725 Biology of the Cell – Lecture 6 – 2024/25 32 Questions?