RNA synth and proc_F23.pdf

RNA Synthesis and Processing – Ch 31 CMB 704/DENT 604 – Fundamental Biochemistry Dr. Maryam Syed [email protected] Fall 2023 1 Learning Objectives • Identify the major types of cellular RNA and the function of each. • Describe the major steps in eukaryotic and prokaryotic transcription of an RNA m...

RNA Synthesis and Processing – Ch 31 CMB 704/DENT 604 – Fundamental Biochemistry Dr. Maryam Syed [email protected] Fall 2023 1 Learning Objectives • Identify the major types of cellular RNA and the function of each. • Describe the major steps in eukaryotic and prokaryotic transcription of an RNA molecule. • Explain the function of the different RNA polymerase enzymes in eukaryotes and prokaryotes. • Describe the major differences between prokaryotic and eukaryotic mRNAs. • Identify the role of the different Transcription Factors in eukaryotes. • Explain the effect of enzymes on chromatin structure that influence gene expression in eukaryotes. • Describe the different posttranscriptional processing and splicing events that occur during the synthesis of eukaryotic rRNAs, tRNAs, and mRNAs. • Explain how small RNAs regulate mRNA expression. 2 Major Topics RNA structures 2. Prokaryotic gene transcription 3. Eukaryotic gene transcription 4. Posttranscriptional modification of RNA 1. 3 noncoding RNA Transcription structural catalytic regulatory 4 Transcription • RNA pol generates single-stranded RNA • Highly selective process • Signals instruct RNA polymerase where to start and stop • Involves regulatory proteins • Transcriptome = all RNA transcripts (coding and noncoding) expressed by genome 5 Structure of RNA Single strand of ribonucleotides RNA has ribose instead of deoxyribose at 2’ carbon RNA has U instead of T rRNA, tRNA, mRNA  important role in translation 6 Ribosomal RNA (rRNA) • 80% of total RNA • Associated with ribosomes • provide sites for protein synthesis • Provide scaffolding for ribosome assembly • Catalytic role: form peptide bonds in protein synthesis • Prokaryotes have 3 species • Eukaryotes have 4 species 7 Transfer RNA (tRNA) • 15% of total RNA • At least 1 tRNA for each of the 20 amino acids (aa) • Contain unusual (modified) bases – modified when? • Extensive interchain base-pairing  2° (cloverleaf) and 3° (L-shaped) structure • Adaptor molecule to carry aa to site of protein synthesis • aa covalently bound to 3’-end • anticodon recognizes genetic code on mRNA 8 Messenger RNA (mRNA) • 5% of total RNA • Coding RNA – direct carrier of genetic information from gene sequence on DNA • Polycistronic = mRNA carries code for more than one gene • • Prokaryotes Monocistronic = mRNA carries code for one gene • Eukaryotes • 5’ and 3’ UTR regions • 5’ cap and poly-A tail at 3’-end in eukaryotes (not found in prokaryotes) 9 Prokaryotic Gene Transcription RNA polymerase Steps in RNA synthesis from DNA 10 Prokaryotic RNA Polymerase • One species of RNA pol synthesizes all RNA • exception: Primase synthesizes RNA primers for DNA replication • Multi-subunit enzyme • Synthesizes complementary RNA copy of DNA template • Synthesizes in 5’ to 3’-end • Recognizes promoter region on DNA and unwinds DNA • Only one strand of DNA serves as template for a gene • strand determined by gene promoter 11 Prokaryotic RNA Polymerase • Only one strand of DNA serves as template for transcription • strand determined by gene promoter 12 RNA Polymerase Holoenzyme Prokaryotes • 5 core enzymes: • 2 α and 1 Ω required for enzyme assembly • 1 β’ required for template binding • 1 β required for 5’ to 3’ polymerase activity • σ subunit (aka sigma factor) • Enables recognition of promoter region 13 Transcription in E. Coli • Three phases: 1. Initiation Elongation 3. Termination 2. • Transcription unit = promoter region to termination region • Primary transcript = initial product of transcription 14 Transcription Initiation - Prokaryotes RNA pol binds promoter region on DNA • Promoter region is not transcribed • Promoter contains consensus sequence – 2 conserved regions: Pribnow box: 5′-TATAAT-3′ located 10 bases upstream of transcription start site 1. • Essential to start transcription -35 sequence: 5′-TTGACA-3′ located 35 bases upstream of transcription start site 2. • Allows for high transcription rate 15 Transcription Elongation - Prokaryotes 1. RNA pol continues to unwind DNA 2. RNA pol uses NTPs as substrates (ribonucleotides) 3. Transcript synthesis begins 4. Short transcripts synthesized and discarded 5. Sigma factor released after catalyzing 10 reactions 6. Core enzyme clears promoter and moves along template 7. DNA-RNA hybrid formed 16 Transcription Elongation - Prokaryotes • RNA pol does not require primer • Misincorporation of base  RNA pol pauses, backtracks, cleaves single nt, and restarts • Transcription has higher error rate than replication 17 Transcription Termination - Prokaryotes Termination signal ρ-independent ρ-dependent 18 ρ-Independent Termination • GC-rich region at end of gene • DNA template creates transcript that is selfcomplementary • RNA folds on itself  GC-rich hairpin loop structure • RNA transcript has string of Us at 3’end • • Weakly binds As on DNA template RNA transcript separates from DNA template when DNA double helix reanneals behind RNA pol 19 ρ-dependent Termination • Requires protein ρ (aka rho factor) • ρ protein = hexameric ATPase with helicase activity • ρ binds C-rich rho utilization site (rut) on RNA transcript • ρ moves along RNA until it reaches RNA pol stalled at Terminator • ρ helicase activity separates RNA-DNA hybrid helix to release transcript 20 Eukaryotic Gene Transcription 1. Chromatin structure and gene expression 2. Nuclear RNA pols 21 Eukaryotic gene transcription • More complicated process than in prokaryotes – compartmentalization • Different RNA pols to synthesize rRNA, tRNA, mRNA • Transcription factors (TF) • Bind DNA in core promoter region • Required for assembly of transcription initiation complex • Determine genes for transcription • Each RNA pol has its own promoter regions and TF • Chromatin region to be transcribed must be decondensed 22 Chromatin structure and gene expression – Eukaryotes • Actively transcribed genes are in decondensed form = euchromatin • Inactive DNA is in highly condensed form = heterochromatin • Transition of DNA between euchromatin and heterochromatin = chromatin remodeling • Chromatin remodeling achieved through covalent modifications of histones 23 Chromatin structure and gene expression – Eukaryotes • Acetylation of histone lysine residues at amino terminus by histone acetyltransferase (HAT)  removes (+) charge  decreases histone interaction with (-) charged DNA • Acetyl group provided by Acetyl coenzyme A • Histone deacetylases (HDAC) restores (+) charge on lysine  promotes interaction between histone and DNA Acetylation of histone lysine residues = ? 24 Eukaryotic Nuclear RNA polymerases • 3 types of nuclear RNA pol • Large enzymes with multiple subunits • Each type recognizes specific set of genes 25 RNA Pol I - Eukaryotes • Located in the nucleolus • Synthesizes precursor of mature rRNA 26 RNA Pol II - Eukaryotes • Located in the nucleoplasm • Synthesizes nuclear precursors of mRNA (hnRNA) • Synthesizes small ncRNA • snoRNA • snRNA • miRNA 27 Promoter for RNA Pol II - Eukaryotes • TATA and Core promoter elements • Specifies start site and polarity (which strand is template) • cis-acting regulatory elements – on same DNA that is being transcribed • Sequences are on same DNA as gene being transcribed • Serve as binding sites for general transcription factors (GTF) • GTF interact with each other and RNA pol II 28 Promoter for RNA Pol II - Eukaryotes 1. • TATAAA sequence (similar to Pribnow box) is ~25nt upstream of transcription start site (TSS) = TATA (Hogness) box Some genes do not contain TATA box 2. Core promoter elements • Inr (initiator) located +1 of TSS • DPE (downstream promoter element) located +25 of TSS 29 General Transcription Factors Eukaryotes • RNA pol II cannot recognize and bind directly to promoter • GTF required to: 1. Recognize promoter 2. Recruit RNA pol II to promoter 3. Form preintiation complex 4. Initiate transcription • GTFs are trans-acting elements • Transcribed from different gene then come to site of action 30 General Transcription Factors Eukaryotes TFIID 1. • • Contains TATA-binding protein and TATA-associated factors Recognizes and binds TATA-box and other core promoter elements TFIIF 2. • Recruits RNA pol II to promoter TFIIH 3. • • Helicase activity melts DNA to separate strands at TSS Kinase activity phosphorylates RNA pol II  RNA pol II clears promoter and continue transcription 31 Regulatory Elements and Transcriptional Activators - Eukaryotes • Additional consensus sequences upstream of core promoter • Influence rate of formation of the transcription initiation complex • Consensus sequence within 200 nt of core promoter = proximal regulatory elements • • i.e. CAAT and GC boxes Consensus sequence further than 200 nt from core promoter = distal regulatory elements • i.e. enhancers 32 Regulatory Elements and Transcriptional Activators – Eukaryotes • Specific Transcription Factors (STF; transcriptional activators) bind proximal and distal regulatory elements • STF binding to proximal element • • Regulates frequency of transcription initiation STF binding to distal element • • Mediates response to signals (hormones) Regulates which genes are expressed 33 Regulatory Elements and Transcriptional Activators – Eukaryotes • STF has 2 binding domains 1. DNA-binding domain 2. Transcription activation domain • • • Recruits GTF to core promoter Recruits coactivator proteins (HAT enzymes) involved in chromatin modification Mediator (multisubunit coactivator) • Binds RNA pol II + GTF + STF to regulate transcription initiation 34 Role of Enhancers – Eukaryotes • Special DNA sequences • Increase rate of transcription initiation by RNA pol II • Located on same chromosome as gene to be transcribed 1. 2. 3. Upstream or downstream of transcription start site Close to or very far from promoter On either DNA strand 35 Role of Enhancers – Eukaryotes • Contain response element that binds STF • Enhancer sequence bends or loops DNA for STF to interact with other TF bound to promoter and RNA pol II to stimulate transcription • Silencers (similar MOA as enhancers) function to reduce gene expression 36 RNA Pol III – Eukaryotes • Located in the nucleoplasm • Synthesizes tRNA, 5S rRNA, and some snRNA and snoRNA 37 Posttranscriptional modifications of RNA rRNA tRNA Eukayotic mRNA 38 Posttranscriptional Modification of RNA • First, linear, RNA copy of transcription unit = primary transcript • Primary transcript of rRNA and tRNA in euk and prok is modified posttranscriptionally by ribonuclesases • Cleave original transcript • Further posttranscriptional modifications in tRNA gives each species a unique identity • Prok mRNA does not usually undergo posttranscriptional modifications • Euk mRNA does undergo posttranscriptional modifications 39 Posttranscriptional modifications of rRNA • rRNA (prok and euk) generated from long pre-rRNA transcript • 23S, 16S, and 5S rRNA of prokaryotes produced from a single pre-rRNA transcript • 28S, 18S, and 5.8S rRNA of eukaryotes produced from a single pre-rRNA transcript 40 Posttranscriptional modifications of rRNA 1. Pre-rRNA are cleaved by ribonucleases  intermediatesized pieces of rRNA, 2. Intermediate rRNA further processed (trimmed by exonucleases and modified at some bases and riboses)  produce required RNA species • Euk rRNA synthesis and processing occurs in nucleolus • Base and sugar modifications facilitated by snoRNA 41 Posttranscriptional modifications of tRNA • tRNA (prok and euk) generated from long pre-tRNA transcript • Sequences at 5’ and 3’ end removed • Introns removed from anticodon loop by nucleases • -CCA sequence added by nucleotidyltransferase to 3’-terminus of tRNA • Bases at specific positions modified  unusual bases 42 Posttranscriptional modifications of Eukaryotic mRNA • Primary transcripts synthesized by nuclear RNA pol II = heterogeneous nuclear RNA (hnRNA) • Pre-mRNA of hnRNA undergo extensive modifications 1. Addition of 5’-cap Addition of 3’-poly-A tail 3. Splicing 4. Alternative splicing 2. 43 Eukaryotic mRNA: Addition of 5’-cap • First modification of pre-mRNA • 5’-cap stabilizes mRNA for initiation of translation • 5’-cap = 7-methylguanosine attached to the 5′-terminal end of the mRNA • makes the mRNA resistant to most nucleases • S-Adenosylmethionine is source of methyl group 44 Eukaryotic mRNA: Addition of a 3′-poly-A tail • Most mature mRNA have chain of 40–250 adenylates (adenosine monophosphates) attached to the 3′-end • Added by nuclear polyadenylate polymerase (uses ATP as substrate) 1. Pre-mRNA cleaved downstream of polyadenylation signal sequence (AAUAAA) at 3’end 2. Poly-A tail is added to the new 3′-end 3. Signals termination of euk transcription 4. Mature mRNA enters cytosol where poly-A tail is gradually shortened 45 Eukaryotic mRNA: Splicing Removal of introns and intervening sequences from pre-mRNA 1. • Joining of exons 2. • • Non-protein coding sequences Protein-coding sequences Primary mRNA transcripts can have: • • • No introns Little introns Many introns 46 Eukaryotic mRNA: Splicing Role of small nuclear RNA • Uracil-rich snRNA + other proteins form 5 small nuclear ribonucleoprotein particles = snRNP (U1, U2, U4, U5, U6)  mediate splicing • snRNP facilitate removal of introns by forming base pairs with consensus sequences at each end of the intron 47 Eukaryotic mRNA: Alternative Splicing • >90% or pre-mRNA can be spliced in alternative ways in different tissues • Diverse set of proteins produced from limited set of genes • mRNA undergoes tissue-specific alternative splicing to produce multiple isoforms of a protein 48 Summary I • Transcription is the synthesis of RNA from a DNA template. • The enzyme RNA polymerase transcribes genes into a single-stranded RNA. • The RNA produced is complementary to one of the strands of DNA, which is known as the template strand. The other DNA strand is the coding, or sense, strand. • Bacteria contain a single RNA polymerase; eukaryotic cells use three different RNA polymerases. • The DNA template is copied in the 3′-to-5′ direction and the RNA transcript is synthesized in the 5′-to-3′ direction. • In contrast to DNA polymerases, RNA polymerases do not require a primer to initiate transcription, nor do they contain extensive error-checking capabilities. 49 Summary II • Promoter regions, specific sequences in DNA, determine where on the DNA template RNA polymerase binds to initiate transcription. • Transcription initiation requires several protein factors to allow for efficient RNA polymerase binding to the promoter. • Other DNA sequences, such as promoter-proximal elements and enhancers, affect the rate of transcription initiation through the interactions of DNA-binding proteins with RNA polymerase and other initiation factors. • Eukaryotic genes contain exons and introns. Exons specify the coding region of proteins, whereas introns have no coding function. • The primary transcript of eukaryotic genes is modified to remove the introns (splicing) before a final, mature mRNA is produced. 50