BCH3033 Biochemistry 1 Chapter 26 PDF

BCH3033: Biochemistry 1 Chapter 26 04.09.2024 Donella Beckwith, Ph.D. [email protected] 1 Transcription Produces RNA Molecules from DNA transcription = process by which an enzyme system converts the genetic information in dsDNA (double stranded) into an RNA strand with a complementary base sequence...

BCH3033: Biochemistry 1 Chapter 26 04.09.2024 Donella Beckwith, Ph.D. [email protected] 1 Transcription Produces RNA Molecules from DNA transcription = process by which an enzyme system converts the genetic information in dsDNA (double stranded) into an RNA strand with a complementary base sequence 2 By Kelvinsong - Own work, CC BY 3.0, https://commons.wikimedia.org/w/index.php?curid=23086203 3 The Cellular Transcriptome the entire chromosome is usually copied during replication transcription is more selective transcriptome = the sum of all the RNA molecules produced in a cell under a given set of conditions – ~76% of the human genome is transcribed into RNA most products are ncRNAs (no apparent protein coding role) only ~2% are coding RNA (mRNA) 4 Major Types of RNA Molecules messenger RNAs (mRNAs) = encode the amino acid sequences of polypeptides (work during the conversion of genetic information in DNA into protein) transfer RNAs (tRNAs) = read the mRNA and transfer the appropriate amino acid to a growing polypeptide chain during protein synthesis (Ch. 27) ribosomal RNAs (rRNAs) = constituents of ribosomes, the cellular machines that synthesize proteins, responsible for conversion of the codes carried by mRNAs into amino acid chains via tRNA noncoding RNAs (ncRNAs) = have a variety of catalytic, structural, and regulatory functions (non-protein coding roles: not translated into a proteins but are involved in biological processes and disease 5 pathogenesis but many functions are unknown) Question 1 Which statement about transcription is false? A. B. C. D. Transcription uses a DNA strand as the template. Transcription is more selective than replication. Most RNA molecules in the transcriptome code for proteins. The majority of the human genome is transcribed into RNA. 6 Response Which statement about transcription is false? C. Most RNA molecules in the transcriptome code for proteins. Most products of transcription are noncoding RNAs (ncRNAs). 7 Critical Question Describe the main differences between replication and transcription? – – – Discuss the products Discuss the purpose Discuss the process 8 RNA Is Synthesized by RNA Polymerases DNA-dependent RNA polymerase = catalyzes transcription – requires a DNA template, four ribonucleoside 5′-triphosphates (ATP, GTP, UTP, and CTP), and Mg2+ RNA polymerase adds ribonucleotide units to the 3′-OH end, building RNA in the 5′→3′ direction (similar to DNA polymerase mechanism) 9 Transcription by RNA Polymerase in E. coli the 3′-OH acts as a nucleophile, attacking the α phosphate of the incoming ribonucleoside triphosphate each nucleotide in the RNA is selected by WatsonCrick base-pairing interaction the overall reaction is: (NMP)n + NTP → (NMP)n+1 + PPi 10 Question 2 What metal plays an important role in transcription, a role similar to phosphoryl group transfer? A. B. C. D. E. manganese iron copper magnesium cobalt 11 Response What metal plays an important role in transcription, a role similar to phosphoryl group transfer? D. magnesium Mg2+ stabilizes deprotonated oxygen atoms of phosphoryl groups and of carboxyl groups on Asp residues within the active site. 12 Question 3 E. coli RNA polymerase does NOT require: A. B. C. D. a template DNA strand. ATP, GTP, UTP, and CTP. two Mg2+. a Mg2+ and Mn2+. 13 Response E. coli RNA polymerase does NOT require: D. a Mg2+ and Mn2+. DNA-dependent RNA polymerase requires a DNA template, all four ribonucleoside 5-triphosphates (ATP, GTP, UTP, and CTP) as precursors of the nucleotide units of RNA, and two Mg2+ ions. 14 Initiation and Elongation of Transcription initiation occurs when RNA polymerase binds at promoter sequences Step 1 – specific sequences in DNA that RNA polymerase binds – direct transcription of adjacent segments of DNA Step 2 – extend b/w -70 and +30 bp from start site (E. coli) – primers are not required during elongation, the growing RNA strand temporarily basepairs with the DNA template to form a short hybrid RNA-DNA Step 3 double helix 3’ 5’ Nontemplate 3’ 5’ 3’ 5’ 5’ Template 3’ 15 Movement of RNA Polymerase Creates Supercoils a transcription “bubble” forms when the DNA duplex unwinds RNA polymerase generates positive supercoils ahead of the “bubble” and negative supercoils behind – due to restricted DNA strand rotation – relieved through topoisomerases (in cell, Ch. 24) “bubble” 16 Template and Nontemplate Strand Nontemplate 5’ 3’ 5’ Template 3’ template strand = DNA strand that serves as template for RNA synthesis nontemplate strand (coding strand) = DNA strand that is identical in base sequence to the transcribed RNA, with U in RNA in place of T in DNA 17 Question 4 Other than having U in the RNA in place of T in the DNA, the base sequence of transcribed RNA: A. B. C. D. is identical to the template DNA strand sequence. is identical to the nontemplate DNA strand sequence. is the reverse of the template DNA strand sequence. is the reverse of the nontemplate DNA strand sequence. 18 Response Other than having U in the RNA in place of T in the DNA, the base sequence of transcribed RNA: B. is identical to the nontemplate DNA strand sequence. The strand that serves as template for RNA synthesis is called the template strand. The DNA strand complementary to the template, the nontemplate strand, or coding strand, is identical in base sequence to the RNA transcribed from the gene, with U in the RNA in place of T in the DNA. 19 Six Subunits Constitute the RNA Polymerase Holoenzyme the σ subunit directs the enzyme to specific DNA binding sites – has variants designated by molecular weight – the most common subunit is σ70 but there are others RNA polymerases lack a separate proofreading 3′→5′ exonuclease active site – leads to a higher error rate What does the 70 stand for? Do DNA polymerases have proofreading? E. coli 20 Question 5 Which subunit is NOT part of the E. coli DNA-dependent RNA polymerase holoenzyme? A. B. C. D. E. α subunit β subunit γ subunit σ subunit ω subunit 21 Response Which subunit is NOT part of the E. coli DNA-dependent RNA polymerase holoenzyme? C. γ subunit The DNA-dependent RNA polymerase of E. coli is a large, complex enzyme with five core subunits (α2ββ′ω) and a sixth subunit, one of a group designated σ. These six subunits constitute the RNA polymerase holoenzyme. 22 Consensus Sequences and the UP Element consensus sequence = formed by certain nucleotides that are particularly common at a given position 1. (5′)TATAAT(3′) at the -10 region (E. coli) 2. (5′)TTGACA(3′) at the -35 region (E. coli) UP (upstream promoter) element = a third AT-rich recognition element that occurs between positions -40 and -60 in the promoters of certain highly expressed genes (highly specific so we won’t be going into detail on this) – bound by the α subunit of RNA polymerase 23 Promoter Recognition by σ70 RNA Polymerase Holoenzyme What does the +1 mean? 24 E. coli The Footprinting Technique is a Way to Find a DNA-Binding Site footprinting = identifies the DNA sequences bound by a particular protein derived from sequencing transcription regulation has been extensively studied using this method 25 Question 6 What valuable piece of information is obtained by DNA footprinting? A. the location of a promoter region of DNA B. the specific sequence of bases to which a particular protein binds C. the location of the 5′ end of a coding sequence D. all the DNA sequences in a genome to which a known DNA sequence binds E. the location(s) in genomic DNA to which a known sequence of RNA binds 26 Response What valuable piece of information is obtained by DNA footprinting? B. the specific sequence of bases to which a particular protein binds Footprinting, a technique derived from principles used in DNA sequencing, identifies the DNA sequences bound by a particular protein. 27 *Initiation and Elongation by E. coli RNA Polymerase* Step 3 Step 1 Step 2 28 Step 2 The σ32 Subunit the σ32 subunit = specific for the heat shock promoters – RNA polymerase binds these promoters only when σ70 is replaced with σ32 the products of heat shock genes are made at higher levels when the cell is exposed to environmental stress – Increase in temperature using different σ subunits allows the cell to coordinate the expression of sets of genes, permits major changes in cell physiology Cytoprotective mechanism which protects neural cells from various stresses (hypothermia, ischemia, …) 29 Question 7 When does the σ subunit dissociate from the RNA polymerase core subunits? A. randomly as the polymerase enters the elongation phase of transcription B. when NusA displaces it C. when the promoter region has passed through D. exactly when the second NTP has been added to the chain 30 Response When does the σ subunit dissociate from the RNA polymerase core subunits? A. randomly as the polymerase enters the elongation phase of transcription The σ subunit dissociates at random as the polymerase enters the elongation phase of transcription. The protein NusA binds to the elongating RNA polymerase, competitively with the σ subunit. 31 Transcription Is Regulated at Several Levels regulation can occur at any step of transcription as the first committed steps in transcription, binding of RNA polymerase to the promoter and initiation of transcription are closely regulated protein binding can activate or repress transcription – cAMP receptor protein (CRP) activates transcription – repressors block RNA synthesis at specific genes 32 Question 8 Signal transduction systems incorporate molecules that have other cellular functions. What second messenger ion or molecule plays a role in activating bacterial gene transcription? A. B. C. D. E. cGMP inositol 1,4,5-trisphosphate 1,2-diacylglycerol Ca2+ cAMP 33 Response Signal transduction systems incorporate molecules that have other cellular functions. What second messenger ion or molecule plays a role in activating bacterial gene transcription? E. cAMP In E. coli, the cAMP receptor protein (CRP) increases the transcription of genes coding for enzymes that metabolize sugars other than glucose when cells are grown in the absence of glucose. 34 Step 3 Specific Sequences Signal Termination of RNA Synthesis ρ-independent terminators (rho): – have a self-complementary region that forms a hairpin – have a conserved string of three A residues that are transcribed into U residues near the 3′ end of the hairpin – polymerase arrives at a terminations site with this structure, it pauses – Facilitates dissociation of the transcript 35 Step 3 ρ-Dependent Terminators ρ (rho) = protein factor that has an ATP-dependent RNA-DNA helicase activity (migrates in the 5’ – 3’ direction) ρ-dependent terminators: class of terminators that rely on the protein factor ρ (rho) – have a CA-rich sequence called a rut (rho utilization) element – ρ promotes release of the RNA (hydrolyzes ATP) – Mechanism of release is not known 36 Question 9 Which factor is NOT associated with the process of transcriptional termination? A. sequence-dependent binding of termination protein in eukaryotes B. dephosphorylation of the CTD of Pol II in eukaryotes C. the action of ρ helicase in E. coli D. hairpin formation in the RNA product of E. coli RNA polymerase 37 Response Which factor is NOT associated with the process of transcriptional termination? A. sequence-dependent binding of termination protein in eukaryotes In eukaryotes, termination begins when elongation factors dissociate. The Pol II carboxyl-terminal domain (CTD) is dephosphorylated, a process facilitated by termination factors. The transcription machinery is then recycled, ready to initiate another transcript. 38 Eukaryotic Cells Have Three Kinds of Nuclear RNA Polymerases eukaryotes have three nuclear RNA polymerases: I, II, and III – each has a specific function and is recruited to a specific promoter sequence Table 26-1 Eukaryotic Nuclear RNA Polymerases RNA polymerase Types of RNA synthesized I Pre-ribosomal RNA II mRNA ncRNA III tRNA 5S rRNA ncRNA 39 RNA Polymerase I (Pol I) RNA polymerase I (Pol I) = responsible for the synthesis of a transcript called pre-ribosomal RNA (or pre-rRNA) pre-rRNA contains the precursor for the 18S, 5.8S, and 28S rRNAs 40 RNA Polymerase II (Pol II) RNA polymerase II (Pol II) = responsible for the synthesis of mRNAs and many ncRNAs – can recognize 1000s of promoters some Pol II promoters have sequences in common: – a TATA box consensus sequence (eukaryotic) (TATA(A/T)A(A/T)(A/G)) near -30 – an Inr sequence (initiator) at +1 – such promoters are in the minority 41 RNA Polymerase III (Pol III) RNA polymerase III (Pol III) = responsible for the synthesis of tRNAs, only 5S rRNA, and other ncRNAs Pol III promoters are well characterized 42 Question 10 Which eukaryotic RNA polymerase is responsible for transcribing MOST rRNAs? A. B. C. D. Pol I Pol II Pol III Pol I and Pol II 43 Response Which eukaryotic RNA polymerase is responsible for transcribing MOST rRNAs? A. Pol I RNA polymerase I (Pol I) is responsible for the synthesis of only one type of RNA, a transcript called pre-ribosomal RNA (or pre-rRNA), which contains the precursor for the 18S, 5.8S, and 28S rRNAs. 44 RNA Polymerase II Requires Many Other Protein Factors for Its Activity Pol II is more complex than its bacterial counterpart, but structure, function, and mechanism are conserved – RBP1 subunit exhibits a high degree of homology to the bacterial β′ subunit – RBP2 subunit is structurally similar to the bacterial β subunit – RBP3 and RBP11 subunits show some structural homology to the two bacterial α subunits 45 The Carboxyl-Terminal Domain (CTD) carboxyl-terminal domain (CTD) = a long carboxylterminal tail in RBP1 that consists of many repeats of a consensus heptad amino acid sequence, -YSPTSPS– yeast enzyme contains 26 repeats – human and mouse enzymes contain 52 repeats – separated from the main body of the enzyme by an intrinsically disordered linker sequence 46 Pol II Requires Other Proteins transcription factors = an array of proteins that work with Pol II form the active transcription complex general transcription factors = those required at every Pol II promoter – Usually designated by TFII and an additional identifier 47 https://www.nature.com/articles/nrm3098 Proteins Required for Initiation of Transcription at Pol II Promoters of Eukaryotes 48 Transcription at RNA Polymerase II Promoters 49 Question 11 Which statement about general transcription factors is false? A. They are required at every Pol II promoter. B. They are usually designated TFII with an additional identifier. C. They are highly conserved in all eukaryotes. D. They are proteins that block the synthesis of RNA at specific genes. 50 ***Response Which statement about general transcription factors is false? D. They are proteins that block the synthesis of RNA at specific genes. The general transcription factors required at every Pol II promoter (factors usually designated TFII with an additional identifier) are highly conserved in all eukaryotes. Repressors are proteins that block the synthesis of RNA at specific genes. 51