CMB 31-32 RNA Structure and Transcription BL 2023.pptx

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RNA Structure & Transcription Bindong Liu, PhD Professor, Microbiology, Immunology and Physiology Meharry Medical College Email: [email protected]; Tel: 615-327-6877 September 2023 Lecture Objectives At the end of this lecture, students should be able to: • Describe the “flow of genetic information” •...

RNA Structure & Transcription Bindong Liu, PhD Professor, Microbiology, Immunology and Physiology Meharry Medical College Email: [email protected]; Tel: 615-327-6877 September 2023 Lecture Objectives At the end of this lecture, students should be able to: • Describe the “flow of genetic information” • Describe the types of RNA and their structure • Describe the process of RNA transcription and its important components • Describe mRNA processing Flow of Genetic Information • Central dogma of Molecular Biology DNA → RNA → Protein • All living cells • Transcription: DNA → RNA • Translation: RNA → protein Molecular Biology of the Cell (© Garland Science 2015) The Central Dogma in prokaryotic versus eukaryotic cells • In prokaryotes  no nucleus  all three processes occur simultaneously • In eukaryotes  with a nucleus  in nucleus DNA replication RNA transcription and post-transcription processing  in cytoplasm translation RNA vs. DNA RNA DNA • Single stranded • Ribose sugar • Double stranded • Deoxyribose sugar • Bases: C,G,A,U • Bases: C,G A,T Both contain a sugar, phosphate, and base. RNA can fold into specific structure • RNA often forms conventional base pairs (red) with complementary sequences and unconventional base pairs (green), such as G:U • the ability to fold into complex threedimensional shapes allows some RNA molecules to have precise structural and unique functions. Types of RNA  Messenger RNA (mRNA) Long strands of RNA nucleotides that are formed complementary to one strand of DNA code for proteins  Ribosomal RNA (rRNA) Associates with proteins to form ribosomes in the cytoplasm  Transfer RNA (tRNA) Smaller segments of RNA nucleotides transport amino acids to the ribosome, where proteins are made by adding one amino acid at a time Coding vs. Non-coding RNA • Coding RNA, mRNA: coding protein • Non-coding RNA (ncRNA): do not code protein • ncRNAs serve as enzymatic, structural, and regulatory components for a wide variety of processes in the cell. • RNA makes up a few percent of a cell’s dry weight • Most of the RNA is rRNA (~80% of total RNA) • mRNA comprise 3-5% of total RNA Transcription Main Steps: • A small portion of the DNA double helix opens and unwinds to expose bases on each DNA strand • One of the two strands then acts as a template for the synthesis of an RNA molecule • The nucleotide sequence of the RNA chain is determined by complementary basepairing between incoming nucleotide and DNA • Incoming ribonucleotide is covalently linked to the growing RNA chain in an enzymatically catalyzed reaction • The RNA chain produced by transcription— the transcript Transcription • The enzymes that perform transcription are called RNA polymerases • ATP, CTP, UTP and GTP are the substrates • RNA polymerase catalyzes sugar-phosphate bond between 3´-OH of ribose & 5´-PO4 • The hydrolysis of the high-energy bonds provides the energy for the reaction • New RNA strand is immediately released from DNA - many RNA copies can be made from the same gene in a relatively short time Transcription: RNA Polymerase • DNA-dependent DNA template, ribonucleoside 5´ triphosphates, and Mg2+ • No primer is needed • Synthesizes RNA in 5´ to 3´ direction • E. coli only has one RNA polymerase • Eukaryotes have three RNA polymerases r m t Remember: I, II, III => r. m. t RNA Polymerase vs. DNA Polymerase Molecular Biology of the Cell (© Garland Science 2015) • RNA polymerase catalyzes the linkage of ribonucleotides, not deoxyribonucleotides • RNA polymerases can start an RNA chain without a primer • DNA polymerases make their products in segments • RNA polymerases finish making the entire RNA • RNA polymerase error rate: 1 in104 nucleotides • DNA polymerase error rate: 1 in 107 nucleotides • The higher error rate is acceptable since RNA does not permanently store genetic information in cells • RNA polymerase has a modest proofreading mechanism Stages of Transcription • Promoter recognition • Chain initiation • Chain elongation • Chain termination • Works differently in bacteria and Eukaryotes Transcription Cycle of Bacterial RNA Polymerase • Sigma (σ) factor binds the core enzyme to form RNA polymerase holoenzyme • The holoenzyme binds to the promoter due to the σ factor making specific contact with the DNA helix Scrunching mechanism • The holoenzyme partially opens the dsDNA (about 10 nucleotides), called transcription bubble • One strand is stabilized by the σ factor, and the other strand is used as a template for RNA synthesis • Molecular Biology of the Cell (© Garland Science 2015 The first ten or so nucleotides are synthesized by the scrunching mechanism repeatedly and released • The core enzyme breaks free of the promoter, discards the σ factor, and begins to move down the DNA to synthesize RNA stepwise A Few Terms • Promoter- a region of DNA that initiates transcription of a particular gene • Transcription factors o Proteins involved in transcribing DNA into RNA, to position eukaryotic RNA polymerase, aid in pulling apart the two strands of DNA to allow transcription to begin, and release RNA polymerase from the promoter to start its elongation mode. o General/Basal transcription factors are proteins needed at nearly all promoters used by RNA polymerase II. TFIIA, TFIIB, TFIIC, TFIID, and so on. • Elongation factors - are proteins that decrease the likelihood that RNA polymerase will dissociate before it reaches the end of a gene. General/Basal Transcription Factors TBP: TATA-binding protein TAF: TBP-associated factors Consensus sequences found in the vicinity of eukaryotic RNA polymerase II start points • Four core promoter elements • TATA box locate ~25bp upstream of INR • Usually, only two to three of them present in transcription • Most of them are located upstream of INR • DPE is downstream of INR Transcription: 1. Promoter Recognition • Transcription factors bind to the promoter and recruit RNA pol to form transcription initiation complex • TFIIH, containing DNA helicase activity, unwinding a 12–15 bp segment of the DNA • TFIIH phosphorates the CTD of RNA pol II, which enables pol II to disengage from a cluster of general transcription factors • Pol II acquires new proteins for long distances transcription • Once elongation starts, most of the general transcription factors are released from the DNA Molecular Biology of the Cell (© Garland Science 2015) Transcription: 2. Transcription Initiation • Polymerase II also requires activator, mediator, and chromatinmodifying proteins • Transcription activator binds to enhancer to attract RNA pol II to the start point • Mediator is required to allow the activator to properly communicate with pol II and general transcription factors Molecular Biology of the Cell (© Garland Science 2015) • Chromatin-modifying enzymes are required to facilitate the assembly of the transcription initiation machinery onto DNA Transcription: 3. Chain Elongation • RNA polymerase moves along the transcribed or template DNA strand • Requires elongation factors- proteins that decrease the likelihood that RNA polymerase will dissociate before it reaches the end of a gene. • Chromatin remodeling complexes are required to help RNA polymerase move along the chromatin • The new RNA molecule (primary transcript) forms a short RNA-DNA hybrid molecule with the DNA template • Challenges – DNA supercoiling (both in bacteria and eukaryotes) Transcription Creates Superhelical Tension – DNA Supercoiling Molecular Biology of the Cell (© Garland Science 2015) • DNA supercoiling is a barrier to elongating RNA polymerase • A moving polymerase generates positive superhelical tension in the DNA front of it and negative helical tension behind it. • In eukaryotes, DNA topoisomerase removes superhelical tension • In bacteria, DNA gyrase, a DNA topoisomerase, constantly keeps negative supercoils in DNA. When RNA pol opens the DNA helix, it removes these negative supercoils, reducing superhelical tension. Transcription: 4. Chain Termination • Termination is signaled by a sequence that can form a hairpin loop • The polymerase and the new RNA molecule are released upon formation of the loop Prokaryotic and Eukaryotic mRNA Molecules Figure 6-21A Molecular Biology of the Cell (© Garland Science 2008) Figure 6-20 Molecular Biology of the Cell (© Garland Science 2008) RNA processing in eukaryotes • The transcript formed as a result of transcription is NOT a mature RNA – precursor mRNA (pre-mRNA) • It is processed to produce a mature RNA molecule A Eukaryotic Gene Transcription elongation in eukaryotes is tightly coupled to RNA processing • CTD phosphorylation of RNA pol • Movement of RNA pol from the transcription start site allows a new set of proteins to associate with the RNA polymerase tail that functions in transcription elongation and RNA processing. • Some of these processing proteins are thought to “hop” from the polymerase tail onto the nascent RNA molecule to begin processing it as it emerges from the RNA polymerase Molecular Biology of the Cell (© Garland Science 2015) Transcription: mRNA synthesis/processing • Prokaryotes: mRNA transcribed directly from DNA template and used immediately in protein synthesis • Eukaryotes: primary transcript must be processed to produce the mRNA  5´-methylguanosine cap added – First modification that occurs  Noncoding sequences (introns) are removed  Coding sequences (exons) spliced together  3´-polyadenosine tail added • Transcription elongation in eukaryotes is tightly coupled to RNA Processing (CTD phosphorylation) Transcription: mRNA synthesis/processing • RNA splicing - the editing of the pre-mRNA transcript into a mature mRNA. After splicing, introns are removed, and exons are joined together. Transcription: mRNA synthesis/processing • RNA splicing inside the nucleus on particles called spliceosomes • Spliceosomes comprise proteins and small RNA molecules (100– 200 bp; snRNA). • Both proteins and RNA are required, but some research indicates that RNA can catalyze the splicing reaction. • Catalytic RNA: ribozymes • Self-splicing in Tetrahymena: the RNA catalyzes its own splicing • Alternative splicing can produce different forms of a protein from the same gene • Mutations at the splice sites can cause disease – Thalassemia and Breast cancer (BRCA 1) For the RNA sequence 5’ – AUCCGGCCUUAAUUCAUC – 3’, list the sequences of template and coding strands. True or false makes up a few percent of a cell’s dry weight. mRNA is the major component of the tota DNA and RNA contain sugar, phosphate and bases product of transcription is named transcript transcription needs a primer to initiate the process acteria, DNA gyrase constantly keeps positive supercoils in DNA ng a 5’ cap is the first modification that happened to bacterial RNA during RNA processin

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