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Genetic information transfer-----RNA biosynthesis (RNA transcription-转录) Lecturer: Jiao Li Phone: 021-65986142 Email: [email protected] Objectives After completion of this section, you should be able to : 1. Understand RNA polymerase involved in transcription. 2. Describe the process of RNA transcription and post- transcriptional modifications. 4. Appreciate the types of introns and mechanism of splicing. Central dogma replication DNA transcription translation RNA Protein RNA replication reverse transcription Outline of the section 1. Transcription system 2. Biosynthesis process of RNA 3. Post-transcriptional modifications RNA transcription A reaction in which a template strand of DNA is utilized by specific RNA polymerase to generate one kind of RNA. Getting the message out! transcription DNA RNA Types of RNAs RNA type size function Coding RNA: mRNA variable carrying information of protein sequence small carrying amino acid to growing polypeptide Non-coding RNA: Transfer-tRNA Ribosomal-rRNA variable major and active component of ribosome, most abundant RNA Small nuclear-snRNA small RNA processing Small nucleolar-snoRNA small RNA processing Small cytoplasm-scRNA small 7SL-RNA- component of SRP Micro-RNA 21-22nt gene regulation Small interference-siRNA 20-25nt gene regulation Electrophoresis of total RNA from eukaryotes Section1 Transcription system Transcription system Substrates: NTP (ATP, UTP, GTP, CTP) Template: DNA Enzyme: RNA polymerase, RNA-pol Other protein factors §1.1 RNA polymerase- RNA pol a. RNA pol in prokaryote In prokaryotic (bacteria) all types of RNA are synthesized by only one species of RNA polymerase. Subunits of RNA polymerase in prokaryote subunit gene MW α rpo A 40000 β β′ σ ω number 2 rpo B 155000 1 rpo C 160000 1 rpo D 32000-92000 1 9000 function Binding upper promoter element and regulatory factors Polymerase activity Binding to DNA(nonspecifically) Binding specific promoter regions Not known Synthesize RNA Transcription specificity Core enzyme Promoter recognization σ70 : common promoter σ32 : HSP promoter Holoenzyme Rifampicin inhibits DNA-dependent RNA polymerase in bacterial cells Mechanism: by binding its β-subunit of RNA polymerase Bactericidal antibiotic Separate Rifampicin sensitive Rebuild Rifampicin resistance Rifampicin resistance §1.1 RNA polymerase- RNA pol b. RNA pol in eukaryote Sensivity to α-amanitine types RNA pol I function 45S rRNA RNA pol II mRNA many snRNA sensitive RNA pol III tRNA 5S rRNA some snRNA moderately sensitive Mt RNA pol RNA in Mt insensitive “death cap” mushroom CTD: carboxyl terminal domain -, c-terminus of the largest subunit of RNA polⅡ , which consists of repeat sequence-Tyr Ser Pro Thr Ser Pro Ser. Tyr ,Ser and Thr residues in CTD can be phorsphorylated, which is involved in initiation of transcription. RNA polⅡ Characteristics of RNA pol ◦ No primer ◦ No exonuclease activity-no proofreading function §1.2 DNA template • Structural gene : codes for any RNA or protein product. • template strand: antisense strand. Template for guiding RNA synthesis. • coding strand: sense strand. Strand complementary to template strand, same with RNA product. Structural gene 5 3 Transcription direction coding strand template strand template strand coding strand Transcription direction 5′···GCAGTACATGTC ···3′ 3′··· c g t g a t g t a c a g ···5′ Coding strand Template strand Transcription 5′···GCAGUACAUGUC ···3′ mRNA Translation N······Ala · Val · His · Val ······C Protein mRNA: sequence is the same to coding strand. Asymmetric transcription • Asymmetric transcription: for one structural gene, only one DNA strand serves as template for RNA synthesis. • Both DNA strands can serve as template. Specific sequences on DNA are important for transcription RNA pol protection assay RNA pol Ⅱ Digested by nuclease Release of binding fragment It shows RNA pol binding sequence is important for transcription ---- promoter Important sequences for transcription: a. Promoter b. Enhancer Regulatory sequence c. terminator d. Structural gene Promoter Structrual gene Terminator Transcrition unit Operon a. Promoter Promoters：locate near the genes they regulate, on the same strand and typically upstream of the antisense strand. Direct the level and accuracy of transcription of a given gene. Promoter in prokaryote RNA pol binding region Structral gene Promoter in pro： RNA pol 识别和结合 的部位 -35: recognition site -10: binding site +1: initiation site 3 5 5 3 5 3 3 -50 40 30 -35 区 TTGACA AA C T G T (RNA-pol recognition site) 20 10 1 10 5 Initiation site -10 区 T A T A A T Pu A T A T T A Py RNA pol binding site TATA box (Pribnow box) Promoter in eukaryote Cis-acting element in eukaryote-promoter：it is where RNA pol binds by TF. -GCGC---CAAT---TATA structural gene Initiation site TATA box enhancer CAAT box GC box Cis-acting element： specific DNA sequence which can regulate gene expression. Transcription factors –TF：protein which can bind to DNA sequence to control gene transcription. TF types recognized by RNA polⅡ ① General transcription factors : commonly present and interact with promoter region of genes transcribed by RNA polⅡ---TF Ⅱ A,TA Ⅱ B ,TF Ⅱ D ,TF Ⅱ E ,TF Ⅱ F and TF Ⅱ H . ② Upstream transcription factor: recognizing and binding specific sequence of gene surrounding promoter region. ③ Inducible factor: can stimulate or repress gene transcription. b. Terminator Terminator: locates downstream of structural gene and stops transcription. Terminator in prokaryote: form stem-loop structure . Terminator in eukaryote: no 真核生物 mRNA 基因无终止子。 c. Enhancer Section2 Biosynthesis process of RNA §2. RNA transcription process Sequential actions Initiation Elongation Termination §2. 1 Transcription of prokaryotes Initiation 1. Recognize the starting point 2. Separate dsDNA 3. Initiation complex Initiation 1. RNA-pol holoenzyme(2) binds template 2. DNA unwinding 3. Forming initiation complex RNApol (2) - DNA - pppGpN- OH 3 Transcription initiation complex 4. Forming the first phosphodiester bond 5-pppG -OH + NTP 5-pppGpN - OH 3 + ppi G or A Elongation 5. Once the first 10 nucleotides are incorporated, factor dissociates from holoenzyme and the core enzyme left keeps proceeding to extend RNA. 6. RNA product is elongated under catalysis of core enzyme. (NMP) n + NTP (NMP) n+1 + PPi factor dissociate Negative supercoil RNA pol Positive supercoil Direction of transcription Transcription bubble： RNA-pol （core enzyme） ···· DNA ···· RNA Electron micrograph of rRNA transcription “leather” formed during RNA transcription. 5 3 DNA RNA RNA polymerase ribosome Termination Transcription is terminated by signals within the RNA sequence In bacteria, terminators come in two types: Rho-dependent termination Rho-independent termination. Rho-dependent ATP ρfactor：protein, 275 kD . ρ factor can bind ‘C’ rich region in newly synthesized RNA. ρ factor has ATPase and RNA-DNA helicase activity---release RNA from RNA-DNA hybrid double chains. Rho-independent Stem-loop structure in RNA stops RNA pol from elongating. Stem-loop structure Poly A-U in RNA relase RNA from RNA-DNA. A-U §2. 2 Transcription of eukaryotes Differences of transcription between prokaryotes and eukaryotes ① RNA pol in eukaryotes:Ⅰ ,Ⅱ, Ⅲ ② RNA pol Ⅱis directed to promoter by other transcription factors. ③ There are more multiple cis-acting elements in upstream of starting site and more proteins involved in initiation. ④ Termination is coupled with modification. Initiation Transcription factor II（TFII）: TFs which direct RNA pol II to promoter and help to initiate transcription. Factor TFIID Subunit Function TBP, TAFs Identifing and binding TATA box TFIIA Stabilizing binding of TFIID TFIIB Forming ternary complex with A and D TFIIF Binding with RNA pol II TFIIE Recruiting TFIIH TFIIH Helicase, kinase activity •TBP: TATA box binding protein, one subunit of TFIID TAFs: TBP associated proteins. TFII D binds TATA box TFIIA and TFII B join to form ternary complex TFII F binds RNA pol II and join the complex CTD The addition of TFII E and TFII F is the final step TFII H unwind DNA strands and phosphates CTD RNA pol II and TFIIs form pre-initiation complex (PIC),PIC is similar to RNA pol holoenzyme in prokaryote. In vivo, Transcription Initiation Requires Additional Proteins Assembly of the pre-initiation complex in presence of mediator, nucleosome modifiers and remodelers, and transcriptional activators. Elongation nucleosome RNA-Pol Dissemble of nucleosome Direction of transcription Dissociation of nucleosome RNA-Pol Reassembly of nucleosome Reassemble of nucleosome RNA-Pol Termination —— Coupling with the addition of poly A tail. AAAAAAA······ 3 mRNA 3tail Nuclease 3 5 5 AATAAA GTGTGTG Site for polyadenylation RNA-pol 3 Section 3 Posttranscriptional processing Posttranscriptional processing After transcription, the formed immature RNA will undergo modifications to be mature and functional. Processing types: ① Cleavage and Splicing ② Terminal addition ③ Modification ④ RNA editing §3. 1 posttranscriptional processing in prokaryote rRNA methylation Methyl group cleavage §3. 1 posttranscriptional processing in prokaryote tRNA cleave addition modification Cleavage Terminal addition modification §3. 2 posttranscriptional processing in eukaryote rRNA rDNA 18S Intron 5.8S 28S Intron 转录 剪接 18S - rRNA 5.8S和28S-rRNA 45S - rRNA tRNA Modifications Exo and endonuclease （1）Methylation example：A Am （2）Reductive reaction tRNA nt transferase example：U DHU （3）Translocation reaction example：U ψ （4）Deamination example：A I modifications mRNA Types of processing : ① Capping ② Addition of poly A tail ③ Splicing ④ Methylation ⑤ RNA editing ① Capping The addition of 5cap (m7GpppGp —) 5’ to 5’ connection The addition of 5cap 5 pppGp… phosphatase 5 ppGp… Pi pppG Guanosine transferase ppi 3 GpppGp… SAM Methyl group transferase 3 m7GpppGp… Cap O Cap Ⅰ Cap Ⅱ Function: 1. Help to stabilize mRNA 2. Permit initiation of translation (specific, where translation should begin). ② Addition of poly A tail Specific endonuclease cuts at modification site. PolyA polymerase catalyzes without template- untemplated extension. polyA :∽more than 250 nt . Histone: no poly A tail. Polyadenylate polymerase Roberts and Sharps found interrupted genein 1977 separately. R looping Mature mRNA hybrids with DNA ③ Splicing Split gene A eukaryotic gene in which the coding sequence is divided into two or more exons that are interrupted by a number of noncoding intervening sequences (introns). Also known as interrupted gene. A B Coding region: A, B, Cand D C D Non-coding area Exon and Intron • Exon DNA sequence present in final mature RNA product and codes for primary sequence of protein. • Intron DNA sequence not present in final mature RNA product and is removed during splicing process. hnRNA Transcription, capping and tail Splicing mRNA Splicing of mRNA —— Introns are removed and exons are joined. 5’Splicing site Branch site 3’ Splicing site Exon upstream-AGGUAAGU--------------A--------(Py)nNCAGG-exon downstream intron •snRNA（U1,2,4,5,6）+ protein = snRNP ( small nuclear ribonucleoprotein) •snRNP+hnRNA= Splicesome Exon Intron Exon Two steps of transesterification reactions Nucleophilic attack 5’-2’ bond Mechanism of action of mRNA splicing Egg-albumin gene Transcription hnRNA Modification in 5’and 3’ hnRNA splicing Muture mRNA Transcription and posttranscription of egg albumin Alternative splicing U1bind to 5’ end of downstream intron Cross-exon splicing U2 bind to branch site A Alternative splicing of tropomyosin (TM) One gene multiple mRNAs multiple proteins （Isoforms） Faulty splicing can cause disease β: There is a point mutation in exon-intron junction of βhemoglobin chain, leading to abnormal splicing of intronand further diminished or absent synthesis of β-chain protein of hemoglobin. ④ Methylation ⑤ RNA editing Apo B gene in human mRNA（14500 nt） mRNA editing Liver: apo B100 （full protein, mw: 500,000） Intestine: apo B48 （truncated protein, mw: 240,000） RNA editing: a kind of process which changes sequence of RNA transcript by untemplated insertion, deletion or substitution. It can alter amino acid sequence of protein coded which differs from that predicted by DNA sequence. • Differential RNA processing Structure of mature mRNA UTR: untranslated region Function of introns 1. Can be further processed after splicing to generate noncoding RNA molecules. 2. Alternative splicing is widely used to generate multiple proteins from a single gene. 3. Represent mobile genetic elements and may be regarded as examples of selfish DNA. Provide more possibilities for genome! Section 4 RNA replication RNA replication （RNA 复制） RNA virus: Viruses need an RNA-dependent RNA-polymerase (RDRP) to replicate their RNA, but animal cells do not seem to possess a suitable enzyme. ——RNA 复制酶为RNA病毒复制所需 RDRP: no primer and no proofreading function. Plus-stranded RNA viruses（正单链RNA病毒 including retrovirus） poliovirus (脊髓灰质炎病毒) SARS病毒 flaviviruses （虫媒病毒） Negative-stranded RNA viruses（负单链RNA病毒） influenza virus (流感病毒) measles virus, mumps virus, (麻疹，腮腺炎病毒) rabies virus (狂犬病毒) Double-stranded RNA viruses（双链RNA病毒） rotaviruses (轮状病毒-呼肠孤病毒reovirus) Replication of RNA virus Plus-stranded RNA viruses ： the same as mRNA and functions as mRNA . Negative-stranded RNA viruses ： complementary to mRNA and need to be copied to plus-sense mRNA. Double-stranded RNA viruses ： cannot function as mRNA and need RDRP. Retrovirus-逆转录病毒 RNA synthesis in viruses Summary 1. There are 1 major enzyme involved in RNA transcription in prokaryote; while there are 3 major enzymes involved in RNA transcription in eukaryote. 2. The process of RNA transcription: initiation; elongation; termination 3. Types of RNA modification: 5’; 3’; splicing; RNA editing; base modification.
