RNA Function and Transcription PDF

RNA types and functions RNA transcription Resource: Molecular Cell Biology 8th Edition Chapter 5, 8, 9 FOUR NITROGENOUS BASES OF DNA Telomerase RNA core domain Simplest definition of a gene: A unit of DNA that contains the information to specify synthesis of a single...

RNA types and functions RNA transcription Resource: Molecular Cell Biology 8th Edition Chapter 5, 8, 9 FOUR NITROGENOUS BASES OF DNA Telomerase RNA core domain Simplest definition of a gene: A unit of DNA that contains the information to specify synthesis of a single polypeptide chain or functional RNA (e.g. tRNA) Approximately 10,000 long noncoding RNAs have been discovered. ‘Complementarity’ RNA is synthesized 5′→3′, RNA polymerase mov down the template DNA strand in a 3′→5′ direction. Does not require ATP Pyrophosphatase (Ppi) Inorganic phosphate Downstream denotes the direction in which a template DNA strand is transcribed; upstream denotes the opposite direction.  2. Approximately 12–14 base pairs of DNA around the transcription start site on the template strand are separated. This allows the template strand to enter the active site of the enzyme. The active site is where catalysis of phosphodiester bond formation between rNTPs that are complementary to the template strand takes place. The 12–14-base-pair region of melted DNA in the polymerase is known as the transcription bubble. Transcription initiation is considered complete when the first two  Transcription bubble 14-20 nucleotides. 1000-2000 nucleotides per minute at 37°C. If an RNA polymerase does dissociate prematurely, it The elongation complex, comprising RNA polymerase, template cannot DNA, and the nascent RNA strand, is extraordinarily stable. For resume synthesis example, RNA polymerase transcribes the longest known mammalian but must start over gene, containing about 2 million base pairs, without dissociating from again at the the DNA template or releasing the nascent RNA. promoter. Termination signals are typically encoded in DNA, and many function by forming an RNA structure that destabilizes the polymerase’s hold on the RNA During elongation... This structure corresponds to the polymerase molecule in the elongation stage Three Eukaryotic RNA Polymerases Catalyze Formation of Different RNAs The Largest Subunit in RNA Polymerase II Has an Essential Carboxy-Terminal Repeat (CTD) Tyr-Ser-Pro-Thr-Ser-Pro- Ser Phosphorylated CTD: red Nonphosphorylated CTD: gree  Eukaryotic Gene Structure: Gene: the entire nucleic acid sequence that is necessary for the synthesis of a functional gene product (polypeptide or RNA)  Processing of Eukaryotic Pre-mRNA Three major events: 5’ capping 3’ cleavage and polyadenylation RNA splicing The 5’ cap is added to nascent RNAs shortly after transcription Initiation 5’ cap marks the RNA molecule as mRNA precursor and protects it from degredation by RNA- digesting enzymes. Capping is catalyzed by a dimeric capping enzyme, which associates with and is activated by phosphorylated CTD of PolII. Capping is also coupled to elongation of PolII. 5’ capping also leads to release of negative elongation factor (NELF). 5’ Capping of mRNA Signifies an mRNA molecule Protects the 5’ end of mRNA from enzymatic degradation Helps transport of mRNA to the cytoplasm Cap is bound by a protein factor (cap binding complex) required for initiation of translation Three enzymes, acting in succession, perform the capping reaction: one (a phosphatase) removes a phosphate from the 5’ end of the nascent RNA, another (a guanyl transferase) adds a GMP in a reverse linkage (5’ to 5’ instead of 5’ to 3’), and a third (a methyl transferase) adds a methyl group to the 5’ and 3’ UTR: mRNA translation and stability 3’ UTR localization of mRNA to specific regions in cytoplasm  Alternative splicing of mRNA Fibronectin is a long, adhesive protein secreted into the extracellular space that can bind other proteins together. The ∼75-kb fibronectin gene contains multiple exons; splicing of the fibronectin transcript varies by cell type. The EIIIB and EIIIA exons (green) encode binding domains for specific proteins on the surface of fibroblasts. The fibronectin mRNA produced in fibroblasts includes the EIIIA and EIIIB exons, whereas these exons are spliced out of fibronectin mRNA in hepatocytes. this fibronectin isoform adheres fibroblasts to the extracellular matrix. Alternative Consequently, splicing of the fibronectin primary transcript in hepatocytes, the major type of cell in the liver, yields mRNAs that lack the EIIIA and EIIIB exons. As a result, the fibronectin secreted by hepatocytes into the blood does not adhere tightly to fibroblasts or to most other cell types, which allows it to circulate. A Diverse Set of Proteins with Conserved RNA Binding Domains Associate with Pre-mRNAs From the time nascent transcripts first emerge from RNA polymerase II until mature mRNAs are transported into the cytoplasm, the RNA molecules are associated with an abundant set of nuclear proteins. Heterogeneous ribonucleoprotein particles (hnRNPs) contain hnRNA and are involved in further processing of pre-mRNA. hnRNP proteins contain one or more RNA-binding domain as well as at least one domain that interacts with other proteins. Functions of hnRNP Proteins: Increasing the accessibility and uniformity of pre-mRNAs for interaction with other RNA molecules or proteins. Some hnRNP proteins interact with the RNA sequences that specify RNA splicing or cleavage/polyadenylation and contribute to the structure recognized by RNA- processing factors. Some hnRNP proteins function in the export of mRNA from the nucleus to the cytoplasm. Splicing Occurs at Short, Conserved Sequences in Pre-mRNAs via Two Transesterification Reactions For short transcripts, splicing follows cleavage and polyadenylation. For longer transcripts splicing occurs co- transcriptionally. Comparison of genomic DNA vs. cDNA sequences for identification of splice sites (exon-intron junctions). Generally only 30–40 nucleotides at each end of an intron are necessary for splicing to occur at normal rates. Spliceosomes, Assembled from snRNPs and a Pre- mRNA, Carry Out Splicing The five splicing snRNPs and other proteins involved in splicing assemble on a pre-mRNA, forming a large ribonucleoprotein complex called a spliceosome. Average exon length in humans 150 bp Average intron length 3500 bp; longest intron 500 kb! Additional sequences are required in long introns for splicing. SR Proteins (RNA-binding proteins) bind to sequences in exons called exonic splicing enhancers.  Spinal Muscular Atrophy (SMA) Approximately 15 percent of the single-base mutations that cause human genetic diseases interfere with proper exon definition. 3′ Cleavage and Polyadenylation of Pre- mRNAs Are Tightly Coupled symplekin Transcription control regions: promoters and other types of control elements (promoter-proximal elements and distant enhancers) Both types of elements can stimulate transcription even when inverted. Both types are often cell-type-specific. The TATA Box, Initiators, and CpG Islands Function as Promoters in Eukaryotic DNA TATA BOX: If the base pairs between the TATA box and the normal transcription start site are deleted, transcription of the altered, shortened template begins at a new site about 25 bp downstream from Initiator the TATA box. sequences: Alternative to TATA Box. C at -1; A at +1 (5′) Y-Y-A+1-N-T/A-Y-Y-Y (3′) degenerate consensus sequence Nucleotide immediately surrounding the start site determines the strength of promoter.  CpG Islands: Loosely defined initiation site as opposed to TATA box and initiator sequences. Found in many protein-coding genes in mammals (70%). Genes encoding proteins that are not required in large amounts. Several alternative sites within 100-1000 bp of high CG content. These promoters are called CpG islands. CpG islands correspond to nucleosome free regions of DNA.  Divergent Transcription from CpG Island Promoters Divergent transcription generates upstream antisense RNAs near the 5′ end of genes that are typically short (50– 2,000 nucleotides) and relatively unstable.  General Transcription Factors Initiation of transcription by RNA Polymerase II requires several initiation factors. General transcription factors: required at almost all RNA PolII transcribed genes (TFIIA, TFIIB...). TFIID: 38 kDa TATA box binding protein (TBP) and 13 TBP associated factors (TAFs). They position RNA Polymerase II at start sites and assist in initiation  TBP is the first protein to bind to TATA box. TFIIA associates with TBP and DNA upstream of TBP- TATA box complex. C-terminal domain of TFIIB contacts both TBP amd DNA on either side of TATA box.. N-terminal domain of TFIIB is inserted into the RNA exit channel of RNA PolII during initiation. TFIIB N terminal helps PolII melt template DNA TFIIF-PolII complex binds. TFIIE binds and creates a docking site for TFIIH. TFIIH binds and preinitiation complex forms. TBP-TFIID PreInitiation TFIIA Complex: General TFIIB TFs and PolII TFIIF-PolII bound to a TFIIE promoter to TFIIH initiate transcription.  Helicase activity of a TFIIH subunit uses ATP to form an open complex where DNA is melted and the template strand is bound at the polymerase active site. N-terminal domain of TFIIB is released from the RNA exit channel and 5’ end of the nascent RNA enters it. TFIIH kinase module phosphorylates PolII CTD on serine 5 (docking site for cap structure formation). General transcription factors (except for TBP) dissociate. Direction of genes may differ.... Organization of Genes in Prokaryotic and Eukaryotic DNA  Match the following: 1. cytoskeleton...... A. Site for ribosomal RNA synthesis 2. nucleolus..... B. Transcription of 18S rRNA gene 3. Centromere..... C. Transcription of actin messenger RNA 4. RNA Polymerase I..... D. Links a pair of sister chromatids together during cell division 5. RNA Polymerase II..... E. Control movement of structures witin the cell 6. RNA Polymerase III...... F. Transcription of tRNA gene  Match the following: 1. cytoskeleton..E.... A. Site for ribosomal RNA synthesis 2. nucleolus..A... B. Transcription of 18S rRNA gene 3. Centromere..D... C. Transcription of actin messenger RNA 4. RNA Polymerase I..B... D. Links a pair of sister chromatids together during cell division 5. RNA Polymerase II...C.. E. Control movement of structures witin the cell 6. RNA Polymerase III...F... F. Transcription of tRNA gene List the factors that affect the melting temperature (Tm) of DNA. GC content Ion concentration of the solution that the DNA is in Chemicals that destabilize hydrogen bonds (e.g. Urea, formamide) Extreme pH Temperature Match the following: ( ) Two amino acids A. phophodiester bond ( ) Guanine and cytosine B. glycosidic bond ( ) Two glucose molecules C. peptide bond ( ) nucleic acids D. hydrogen bond Match the following: ( C) Two amino acids A. phophodiester bond ( D) Guanine and cytosine B. glycosidic bond ( B) Two glucose molecules C. peptide bond ( A ) nucleic acids D. hydrogen bond The DNA Replication Fork Is Asymmetrical 1. 2. 3. 4. The DNA Replication Fork Is Asymmetrical © 2017 Pearson Education, Ltd. Figure 2-1 © 2017 Pearson Education, Ltd. Figure 2-1