Fundamentals of Gene Expression Transcription I PDF

Transcription I [1] Paul J. McDermott, Ph.D. Office: (843) 792-3462 Email: [email protected] TRANSCRIPTION I A. OVERVIEW OF TRANSCRIPTION 1. Transcription Enzyme: RNA Polymerase 2. Direction of RNA Synthesis 3. Transcription Template 4. de novo Initiation of RNA Synthesis 5. Elongation Reaction of R...

Transcription I [1] Paul J. McDermott, Ph.D. Office: (843) 792-3462 Email: [email protected] TRANSCRIPTION I A. OVERVIEW OF TRANSCRIPTION 1. Transcription Enzyme: RNA Polymerase 2. Direction of RNA Synthesis 3. Transcription Template 4. de novo Initiation of RNA Synthesis 5. Elongation Reaction of RNA Polymerases B. TRANSCRIPTION IN PROKARYOTES 1. RNA Polymerase 2. Gene Promoter 3. Initiation Step of Transcription 4. Elongation Step of Transcription 5. Termination Step of Transcription C. TRANSCRIPTION IN EUKARYOTES 1. Types of RNA Polymerases 2. Basic Components of Eukaryotic Transcription by RNA Polymerases 3. Transcriptional Regulation: cis-Acting Elements & trans-Acting Factors 4. RNA Polymerase I 5. RNA Polymerase II Suggested Reading: Marks’ Basic Medical Biochemistry, 5th Ed: Chapter 14 “Our future understanding of disease and the advance of personalized medicine will benefit from models of human transcriptional regulatory circuitry that integrate information about regulatory sequences and the key transcription factors, cofactors, chromatin regulators and ncRNAs that operate at regulatory sites. The development of these models should thus be among the priorities of biomedical research.” Tong Ihn Lee and Richard A. Young Transcription I [2] OBJECTIVES 1. Specify the 3 main steps of transcription by RNA Polymerases. 2. Contrast the following characteristics between RNA Polymerases and DNA Polymerases: substrate, primers, template, processivity, direction of RNA synthesis, proofreading. 3. Specify the protein subunits of prokaryotic RNA Polymerases. Explain how the sigma (s) subunit is directed to specific promoters to initiate transcription. 4. Differentiate between a closed and open promoter complex. 5. Name the 4 types of eukaryotic RNA Polymerases and specify their corresponding RNA products. 6. Describe the basic components required for eukaryotic RNA Polymerases to initiate transcription of a gene. 7. Differentiate between cis-acting elements and trans-acting factors that regulate transcription of individual genes. 8. Describe the basic components required to form a transcriptionally-active RNA Polymerase I complex and how it functions in the production of ribosomes. 9. Describe the main types of sequence elements in the core promoter of a gene and their corresponding functions in initiation of transcription by RNA Polymerase II. 10. Describe the functions of TFIID, TFIIB, TFIIH and the mediator complex in the initiation step of transcription of all eukaryotic genes. 11. Describe the role of the C-terminal domain of RNA Polymerase II in the elongation step. 12. Explain how the termination step of transcription is linked to polyadenylation of pre-mRNA. Illustrations adapted from: • Biochemistry © 2002 by W.H. Freeman and Company; • The Cell: A Molecular Approach © 2000 ASM Press and Sinauer Associates, Inc. Transcription I [3] A. OVERVIEW OF TRANSCRIPTION DNA [genes] Transcription 5´ 3´ RNA 1. Transcription Enzyme: RNA Polymerase 5´ RNA a) Enzymatic Reaction Mg2+ (RNA)n + NTP (RNA)n+ 1 + PPi NTP = ATP, CTP, UTP, GTP b) Processivity: RNA Polymerases are processive enzymes that transcribe the template strand at rate of 50 nucleotides/sec. template strand of DNA c) Main Steps of Transcription • Initiation • Elongation • Termination d) Absence of Proofreading: RNA Polymerases do not have 3´ to 5´ exonuclease (proofreading) activity 2. Direction of RNA Synthesis RNA is always synthesized in 5´ to 3´ direction. 3. Transcription Template RNA synthesis is template directed. The template strand of DNA is always transcribed in 3´ to 5´ direction by RNA Polymerase. 3´ 4. de novo Initiation of RNA Synthesis 5´ RNA Polymerase initiates RNA synthesis de novo by joining 2 ribonucleotides together to form the first 3´to 5´ phosphodiester bond. Unlike DNA synthesis, a primer is not required. Nucleotide 1 of RNA (usually G or A) CH2 O- Fig 1st phosphodiester bond Nucleotide 2 of RNA O- O- Transcription I [4] A. OVERVIEW OF TRANSCRIPTION 5. Elongation Reaction of RNA Polymerases 3´ 3´ 5´ 5´ RNA RNA PPi 5´ 5´ 3´ 3´ B. TRANSCRIPTION IN PROKARYOTES 1. RNA Polymerase: Prokaryotes have one type of RNA Polymerase Subunit Composition Table Number Function a 2 Binds regulatory proteins with w subunit b 1 Phosphodiester bond formation, Grasps DNA b´ 1 Grasps DNA template s 1 Promoter recognition and initiation s Diagram a2 w b b´ a2 w b b´ s core enzyme holoenzyme s b and b´ form “pincers” that grasp the DNA template during transcription. Transcription I [5] B. TRANSCRIPTION IN PROKARYOTES 2. Gene Promoter: DNA sequence in the template strand of a gene that binds to RNA Polymerase. In prokaryotes, the promoter of most genes has 2 consensus sequences as shown below. Consensus 1 (Pribnow Box) Consensus 2 5´3´- TTGACA AACTGT -36 Start Site TATAAT ATATTA -31 -12 -7 -3´ -5´ +1 Nota Bene: A consensus sequence is derived by determining the base found most frequently at each position in the promoter region of the gene. 3. Initiation Step of Transcription RNA Polymerase holoenzyme binds to DNA nonspecifically. RNA Polymerase searches for promoter site by “sliding” along double-stranded DNA. The s subunit binds specifically to the promoter sequences to form the closed promoter complex. Closed Promoter Complex RNA Polymerase unwinds 17 base pairs of DNA around the initiation site to form the open promoter complex. unwound DNA Open Promoter Complex The first phosphodiester bond is formed during initiation. s subunit is released upon completion of the initiation step. 3´ Elongation by core RNA Polymerase 5´ RNA Transcription I [6] B. TRANSCRIPTION IN PROKARYOTES 3. Initiation Step of Transcription a) Open Promoter Complex Once the s subunit binds specifically to the consensus sequence, helicase activity in the RNA Polymerase holoenzyme unwinds 17 bp of DNA to form an open promoter complex. Fig 5´ 3´ 3´ 5´ RNA Polymerase Holoenzyme b) Functions of the s Subunit • RNA Polymerase holoenzyme is directed to the promoter due to high affinity binding between the s subunit and the promoter sequence. • The s subunit has much lower affinity for non-specific DNA sequences, which enables RNA Polymerase holoenzyme to “slide” along the DNA and find promoter. c) Promoter Specificity • There is only one RNA Polymerase in prokaryotes. However, the RNA Polymerase holoenzyme can initiate transcription of specific genes via the s subunit. • Multiple s subunits exist, each one binds preferentially to a consensus sequence in the promoter of a certain gene. Thus, specificity of the RNA Polymerase for a given promoter is dependent on the s subunit that is part of the holoenzyme. You don’t need to memorize these sequences or specific s subunits, rather, understand the concept that specificity of RNA Polymerase for a promoter is determined by the s subunit. s Subunit s70 Promoter Consensus Sequences -35 TTGACA -10 TATAAT +1 Standard Promoter +1 s32 TNNCNCNCTTGAA Promoter for heat shock genes CCCATNT +1 s54 CTGGGNA TTGCA N = Any Nucleotide Promoter for nitrogenstarvation genes Transcription I [7] B. TRANSCRIPTION IN PROKARYOTES 4. Elongation Step of Transcription Promoter Core RNA Polymerase 3´ s subunit is released upon completion of the initiation step 5´ Elongation 3´ 5´ Fig RNA transcript a) Transcription Bubble The core RNA Polymerase generates a transcription bubble that moves along the chromosome. It consists of the following: • Unwound region = 17 bp of DNA • RNA/DNA duplex = 12 bp Core Polymerase 5´ Neg coil -create -relax b) Role of Topoisomerases The transcription bubble causes overwinding of the DNA ahead of it. To prevent formation of positive supercoils, prokaryotes use Gyrase (Type II Topoisomerase) to create negative supercoils ahead of the bubble. As DNA rewinds, negative supercoils can form. Prokaryotes use Type I Topoisomerases to relax negative supercoils behind the transcription bubble. 5. Termination Step of Transcription a) Template-directed Termination: Sequence in DNA template strand of certain genes contain a G-C rich repeat region followed by a region of A-T base pairs. G-C base-pairing in newly synthesized RNA forms hairpin, which disrupts its association with DNA template strand and halts the transcription reaction. Fxn(2) b) Rho-directed Termination: Rho protein binds to specific sequences in newly synthesized RNA. Rho protein has ATP-dependent helicase activity that dissociates RNA from template strand. Weak bonding between A-U of RNA-DNA duplex causes dissociation of RNA from DNA template. Transcription I [8] C. TRANSCRIPTION IN EUKARYOTES 1. Types of RNA Polymerases Type RNA Products Location RNA Polymerase I 45S Pre-ribosomal RNA [5.8S, 18S, 28S rRNA] Nucleolus RNA Polymerase II Pre-mRNAs, Primary miRNAs snRNAs, lncRNAs Nucleus RNA Polymerase III tRNAs, 5S rRNA, some snRNAs Nucleus Mitochondrial All mitochondrial RNAs Mitochondria 2. Basic Components of Eukaryotic Transcription by RNA Polymerases a) Core RNA Polymerase: Transcription enzyme consisting of multiple protein subunits. b) General Transcription Factors: Proteins required for basal transcription of all genes. c) Specific Transcription Factors: Proteins that regulate transcription of specific target genes. The composition and number of specific transcription factors that regulate each gene differ, depending on its regulatory sequences. The following terminology is often used for specific transcription factors: • Activators (Transactivators) and Repressors (Transrepressors): Regulate transcription by binding directly to DNA regulatory sequences in a gene. They can also bind to other proteins including general transcription factors and mediator proteins. • Mediator Proteins (Coactivators and Corepressors): Do not bind DNA directly, rather they bind to and interact with general and/or specific transcription factors to regulate their activity. Many mediator proteins function in modifying chromatin by epigenetic mechanisms. 3. Transcriptional Regulation: cis-Acting Elements and trans-Acting Factors a) cis-Acting Elements: Regulatory DNA sequences specific to each gene, e.g., Core Promoter, Proximal Promoter Elements, Enhancers, Silencers, Downstream Promoter Elements. b) trans-Acting Factors: Regulatory proteins derived from genes other than the target gene, e.g., General and Specific Transcription Factors (activators, repressors, mediator proteins) Mediator proteins Mediator proteins Core RNA Polymerase Fig Activators Repressor General Transcription Factors +1 Gene cis-Elements: Silencer Enhancer Proximal Element Core Downstream Promoter Element Transcription I [9] C. TRANSCRIPTION IN EUKARYOTES 4. RNA Polymerase I: Enzyme that transcribes rRNA genes in the nucleolus. a) rRNA Genes • A human diploid cell contains approximately 400 rRNA genes arranged in tandem repeats along the 5 acrocentric chromosomes [13, 14,15, 21, 22] in the nucleolus. Each rRNA gene is separated by nontranscribed spacer DNA. Fig • The core promoter for rRNA genes is located in nontranscribed spacer DNA and spans the Core promoter first 167 nt upstream of the transcription start site (+1). tranx Core Promoter Fig rRNA Gene -167 18 S 5.8 S 28 S +1 Transcription by RNA Polymerase I 45S Pre-rRNA • RNA Polymerase I transcribes rRNA genes to generate 45S pre-rRNA. This precursor transcript is processed into 18S, 28S and 5.8S rRNA. • Below: EM of nucleolar chromatin showing 3 tandem repeats of the rRNA gene separated by nontranscribed spacer DNA. Each gene is transcribed by multiple RNA Polymerase I complexes as indicated by the growing array of pre-rRNA transcripts. b) Components of RNA Polymerase I Transcription Complex • Core RNA Polymerase I: Multimeric transcription enzyme consisting of 10-12 subunits • Selectivity Factor 1 (SL1): Complex of 4 general transcription factors that binds to DNA sequence in the core promoter of rRNA gene • Upstream Binding Factor (UBF): Specific transcription factor that binds to DNA sequence in core promoter of rRNA genes and interacts with SL1 to promote assembly of a transcriptionally-active RNA Polymerase I complex RNA Polymerase I Complex Fig SL1 UBF RNA Polymerase I rRNA Gene -167 Core Promoter +1 Transcription I [10] C. TRANSCRIPTION IN EUKARYOTES 5. RNA Polymerase II: Enzyme that transcribes most genes to synthesize mRNA +1 Exon 1 Intron Exon 2 3´ 5´ 5´ Gene 3´ Core Promoter Pre-mRNA (hnRNA) Transcription by RNA Polymerase II 5´ 3´ RNA Processing Mature mRNA Def m7Gppp AAAAAA... a) Core Promoter of Eukaryotic Genes The core promoter of a gene is defined as the minimal stretch of DNA sequence that is sufficient to direct accurate initiation of transcription by RNA polymerase II. The core promoter of most genes ranges between 60-120 bp in length and contains a combination of consensus sequence elements as shown below. Genes vary greatly with respect to types of sequence elements that are present in the core promoter. • Initiator Sequence (Inr): 6 bp sequence that spans the transcription start site (+1) • TATA Box = TATA(A/T)A: Located -25 to -30 bp upstream of the transcription start site • TFIIB Recognition Element (BRE): Approximately -35 bp upstream of transcription start site • Motif 10 Element (MTE): Approximately +18 to +27 downstream of transcription start site • Downstream Promoter Element (DPE): Approximately +28 to +32 downstream of the transcription start site b) Components of RNA Polymerase II Pre-initiation Complex Transcription of all genes by RNA Polymerase II is dependent on assembly of the following components into a pre-initiation complex on the core promoter of the gene. It also is called the basal transcription complex. • Core RNA Polymerase II: Multimeric enzyme consisting of 12 subunits. The C-terminal tail domain (CTD) contains multiple Ser residues that are phosphorylated. • General Transcription Factors (TFs): TFIID, TFIIA, TFIIB, TFIIE, TFIIF, TFIIH. TFIID consists of: TATA box binding protein (TBP) and 13 TBP-associated factors (TAFs). • Mediator: Large coactivator complex that is required for the initiation step of transcription. It consists of approximately 26 proteins that interact with the CTD of RNA Polymerase II, general transcription factors and specific transcription factors. M Scheme fig ed ia to r Schematic of a pre-initiation complex assembled on core promoter of a gene. RNA Specific transcription factors are not Polymerase II shown. The role of specific transcription TAFs factors is to promote assembly of this E complex on the core promoter of H B TBP specific genes to initiate transcription F by RNA Polymerase II above basal A levels. This will be the focus of the TAFs CTD Transcription II lecture. TFIID +1 Exon 1 Transcription I [11] C. TRANSCRIPTION IN EUKARYOTES 5. RNA Polymerase II c) Initiation Step: Role of General Transcription Factors • Initiation of transcription in all genes is dependent on recruitment of RNA Polymerase II to the the core promoter through assembly of a pre-initiation complex with general transcription factors [TFIID, A, B, E, F, H] and the mediator complex. Core promoter of any gene: Sequence elements in the core promoter varies among individual genes. • Current models hold that TFIID directs assembly of the pre-initiation complex, TFIID binds first to the core promoter along with TFIIA. The TBP subunit binds directly to the TATA box if present in the core promoter. However, most genes do not have a TATA box sequence in the core promoter, thus, they are called “TATA-less”. As shown below, TAF subunits function as a “molecular ruler” that positions TBP at the proper DNA binding site in the core promoter. Binding of TFII to core promoter: Note that the TAF subunits form a C-shaped bridge that facilitates interactions between upstream and downstream promoter elements such as MTE and DPE. This ensures that TBP is positioned correctly relative to the transcription start site. TFIIB: Binds to BRE if present in the core promoter. TFIIF & TFIIE: Assembly and stabilization of initiation complex. You don’t need to memorize functions of TFIIF or TFIIE. Me dia to r • TFIIB, TFIIF, TFIIE and TFIIH are recruited along with the core enzyme of RNA polymerase II. This mechanism of sequential assembly ensures correct placement of RNA polymerase II holoenzyme relative to the transcription start site. TFIIH: Composed of multiple subunits for 2 main functions 1) Helicase activity- unwinding DNA strands in the core promoter 2) Protein kinase activity- phosphorylating CTD of RNA Polymerase II, which facilitates promoter release for transcription of template DNA. Mediator: Protein complex required for initiation step by functioning as a link between general and specific transcription factors. Mediator proteins are released from the initiation complex upon phosphorylation of the CTD domain of RNA Polymerase II. d) Elongation Step: Phosphorylation of CTD facilitates release of the RNA Polymerase II core enzyme from the promoter. Additional elongation factors are recruited and activated. e) Termination Step: Termination of transcription is coupled to synthesis of a polyadenylation sequence in the 3´end of pre-mRNA. Cleavage and polyadenylation specificity factor (CPSF) binds to this polyadenylation sequence, which results in downstream cleavage of the 3´-end. An exonuclease is recruited that leads to dissociation of RNA Polymerase II from the template.