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BF Lecture 1 CMB 621 Fogelgren Lecture #1 From DNA to RNA: RNA transcription and mRNA processing Ben Fogelgren, Ph.D. Dept. of Anatomy, Biochemistry, and Physiology John A. Burns School of Medicine University of Hawaii [email protected] phone: 692-1420 (Thanks to Marla Berry and Dulal Borthakur for many slides!) My Objectives (2 lectures): 9/7/17 Chapter 6 of Alberts – Part 1: DNA to RNA • RNA Transcription • RNA Processing • Few Relevant CMB techniques 9/12/17 Chapter 7 - Control of Gene Expression + Article Discussion ** Obviously I can’t compress everything in these chapters into 2 lectures! So, I want you to read these sections, but the lectures will highlight what I think is most important. An overview of the flow information through the cell An overview of the flow information through the cell • A mRNA is made as a complementary copy of the two DNA strands that make up a gene. • The use of mRNA allows the cell to separate information storage from information utilization. • DNA remains stored in the nucleus, while mRNA can be imparted into the cytoplasm for translation. • RNA also allows the cell to greatly amplify its output. • mRNA is translated into proteins by ribosomes, which contain both proteins and RNA. Figure 6-3 Molecular Biology of the Cell (© Garland Science 2008) Gene Transcription • Copying portion of the DNA code to RNA • Enzymes responsible for transcription are called RNA polymerases. • The site on which an RNA polymerase binds prior to initiating transcription is called the promoter. • Cellular RNA polymerases require the help of additional proteins called transcription factors for recognizing promoters. 2 differences between DNA and RNA molecules: (A) Ribonucleotides in RNA (ribose sugar) (B) Uridine (U) instead of Thymidine (T) Classes of RNAs mRNAs, rRNAs, tRNAs snRNAs - small nuclear snoRNAs - small nucleolar tmRNA - transfer/messenger (bacteria) lincRNA – long intergenic non-coding RNAs miRNAs - microRNAs siRNAs - small interfering Lecture #2 shRNAs - short hairpin RNA can form 3D structures • The RNAs of a ribosome are called rRNAs. • tRNAs constitute a third major class of RNA that is required during protein synthesis. • Both rRNAs and tRNAs owe their activity to their complex secondary and tertiary structures. • Many RNAs fold into complex 3-dimensional shapes. • RNA folding is driven by the formation of regions having complementary base pairs. RNA can fold into specific structures Watson-Crick base Non-conventional Structure of an pair interactions base pair interactions actual RNA (Fig. 6.6. Mol Biol of the Cell) Some examples of Non-Watson-Crick or Hoogsteen base pairing found in tRNA Other RNAs in eukaryotic cells • Small nuclear RNAs (snRNAs): Located in the nucleus; involved in RNA splicing, maintaining telomeres etc. • Small nucleolar RNAs (snoRNAs): Small RNA molecules that guide chemical modifications of other RNAs, rRNAs, tRNAs, and snRNAs. • Small interfering RNAs (siRNAs): is a class of double stranded RNAs, 20 -25 nt in length. siRNAs are involved in the RNAi pathway, where they interfere with the expression of a specific gene. • MicroRNAs (miRNAs): single-stranded RNA molecules of 21-23 nt in length. Their main function is to down-regulate gene expression. Our genome encodes hundreds of these tiny microRNAs Chain elongation during transcription The RNA polymerase covers approximately 35 bp of DNA. The transcription bubble contains about 15 bp single-stranded DNA and about 9 bp DNA-RNA hybrid. The enzyme generates overwound (positively supercoiled) DNA ahead of itself and underwound (negatively supercoiled) DNA behind itself. RNA polymerase in the act of transcription elongation The DNA makes a sharp turn in the region of the active site. Transcription Cycle in Bacteria Promoter β’ α σ β α Terminator ω σ factor Core enzyme Holoenzyme β’ α σ β α ω Transcription in bacteria After ~10 nts of RNA synthesis, the core enzyme breaks away its interaction with the promoter and the σ factor. Promoter and terminator sequences are heterogeneous. (Fig. 6.11. Mol Biol of the Cell) Chain elongation Transcription and RNA processing in eukaryotic cells • Eukaryotic cells have three distinct RNA polymerases. • In eukaryotes, a large variety of accessory proteins or transcription factors are also required for transcription. • All three major types of RNAs - mRNAs, rRNAs, and tRNAs - are derived from precursor RNA molecules or primary transcripts. • Primary transcripts have a fleeting existence. Major differences in the transcription machinery between prokaryotes and eukaryotes • While bacteria have only one type of RNA polymerase, eukaryotes have three RNA polymerases • While bacterial RNA polymerase requires only a single σ factor for transcription initiation, eukaryotic RNA requires many additional proteins • Eukaryotic transcription initiation must deal with the packing of DNA into nucleosomes and higher order forms of chromatin structures, features that are absent in bacteria The machinery for mRNA transcription • All eukaryotic mRNA precursors are synthesized by RNA polymerase II. • Initiation of transcription by RNA polymerase II occurs in cooperation with a number of general transcription factors (GTFs). • GTFs are composed of a dozen different subunits, designated as “TFII…”. • GFTs carry out functions similar to σ factor in bacteria. • TFIIF has the same 3-dimentional structure as the equivalent portions of σ factor. The machinery for mRNA transcription • In vast majority of the genes studied, a critical portion of the promoter lies between 24 and 32 bases upstream from the start site of transcription. • This region often contains a consensus sequence 5’TATAAA-3’, which is known as TATA box. • The TATA box of the DNA is the site of assembly of a preinitiation complex that contains the GTFs and the polymerase; this complex must assemble before gene transcription can be initiated. The TATA box 26 bp 25 bp 24 bp Start site of transcription The TATA box is the site of assembly of the preinitiation complex that contains GTFs and the polymerase. Initiation of transcription from a eukaryotic polymerase II promoter • The first step in assembly of the preinitiation complex is binding of a protein, called TATA-binding protein (TBP), that recognizes the TATA box of eukaryotic promoters. • TBP is present as a subunit of a much larger protein complex called TFIID (transcription factor for polymerase II, fraction D). • TFIID also includes a number of other proteins. • TFIID causes a large distortion in the DNA of the TATA box Initiation of transcription from a eukaryotic promoter Start site of trancription TFIID includes the TFIID TBP subunit, which TBP binds to TATA box. TFIID TFIIB TFIIB is thought to provide a binding site for RNA polymerase. Initiation of transcription from a eukaryotic promoter TFIIA binds directly to TBP and TFIIA TFIIH contains stabilizes its binding to DNA 9 subunits, 3 of which possess enzymatic activities. TFIIF contains a subunit homologous TFIIH’s activities to bacterial σ factor include DNA- dependent ATPase helicase, C-terminal domain kinase TFIIE is an α/β heterodimer and it modulates the helicase and kinase activities of TFIIH (Fig. 6.16. Mol Biol of the Cell) Initiation of transcription from a eukaryotic promoter Helicase activity and UTP, ATP, phosphorylation CTP, GTP Disassembly of most GTFs RNA Transcription (Fig. 6.16. Mol Biol of the Cell) The core promoter elements The B recognition TSS element (BRE) is found immediately upstream of the TATA box, and consists of 7 nts G/C G/C G/A C G C C. TFIIB recognizes BRE TATA and binds to it. (-30) BRE Some genes do not (-35) have a TATA box and INR DPE use an initiator (+30) element (Inr) for The downstream promoter element transcription initiation. (DPE) is within the transcribed portion of The Inr element overlaps TSS. a gene INR enhances the strength of a promoter that contains a TATA box. The core promoter elements (Fig. 6.17. Mol Biol of the Cell) TBP bends the DNA molecule approximately 80° and allows TFIIB to bind to the DNA both upstream and downstream of TATA box TATA-binding protein TFIIB TFIIA TFIIB TFIIA BBP TFIID TATA Fig. 6-18 Molecular Biology of the Cell Transcription • Together, RNA polymerase and GTFs are sufficient to promote a low, basal level of transcription from most promoters under in vitro conditions. • Once transcription begins, certain GTFs including TFIID may be left behind at the promoter, while others are released from the complex. • As long as TFIID remains bound to the promoter, additional RNA polymerase molecules may be able to attach and initiate additional rounds to transcription. Multiple transcriptions of genes can occur at same time Gene control region Promoter-proximal element TATA box Enhancer Enhancer Enhancer Enhancer Intron Intron Intron Exon Exon Exon Exon GC box CAAT box Promoter-proximal element includes CCAAT box (or CAAT box) at about 70 bp upstream of +1 and GC box (or GGCG) in a region of 100-150 bp further upstream from the TATA box The CAAT box and GC boxes are also often required for a polymerase to initiate transcription. Besides, CAAT and GC boxes, there may be CpG islands, comprising 20-50 nts CG-rich region within ~100 bp upstream of +1. In mammals, 70 - 80% of CpG cytosines are methylated Ref: Fig. 7.16 Mol Cell Biol, 6th Ed CpG islands ~100 bp upstream Multiple start from the start site sites for transcription Enhancer CpG island CGCGCGCGCG….CGCG Intron Intron Intron Exon Exon Exon Some house-keeping genes that express at low levels have multiple possible transcription start sites over an extended region, 20-200 bp in length. These genes give rise to mRNAs with multiple alternative 5’ ends. They do not have a TATA box or Inr element and their transcription can begin at any of the multiple possible sites within 20-200 bp in length. Most genes of this type contain CpG islands, comprising 20-50 nts CG- rich region within ~100 bp upstream of +1.
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