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Chapter 26 - RNA Metabolism Focus of this chapter is transcription Three main types of RNA (1) Transfer RNA (tRNA) • Carries amino acids to translation machinery • Very stable molecules (2) Ribosomal RNA (rRNA) • Makes up much of the ribosome • Very stable, majority of cellular RNA (3) Messenger RNA (mRNA) • Encodes message from DNA to ribosomes • Rapidly degraded by nucleases There are many other RNAs: - genome of some viruses - specialized catalysts - regulatory functions RNA synthesis Similarities to DNA synthesis -directionality (5'→3‘) -steps- initiation, elongation, termination -copied from a template Differences from DNA synthesis -No primer required -Only discrete segments of DNA template copied -Only one strand of DNA serves as template -Different set of enzymes -Uracil, ribose DNA-dependent RNA Polymerase • RNA polymerase (RNA pol) catalyzes DNAdirected RNA synthesis (transcription) • RNA pol is core of a larger transcription complex • Complex assembles at one end of a gene when transcription is initiated • First nucleotide triphosphate keeps all 3 phosphates • DNA is continuously unwound as RNA pol catalyzes a processive elongation of RNA chain (about 17 bp at a time) transcription bubble • Mechanism of elongation reaction almost identical to that for DNA polymerase • Incoming ribonucleotide triphosphates (RTPs) form correct H bonds to template • New phosphodiester bond formed, PPi released • Adds 50-90 nucleotides/sec (~ 1/10th rate of DNA replication) • Growing RNA molecule “peels off” of DNA as it elongates • DNA reforms double strand Transcription Initiation • Transcription complex assembles at an initiation site (DNA promoter region) • Short stretch of RNA is synthesized • Operon: a transcription unit in which several genes are often cotranscribed in prokaryotes • Eukaryotic genes each have their own promoter Transcription of E. coli ribosomal RNA genes Genes have a 5'→3' Orientation • Convention for double-stranded DNA: Coding strand (non-template)(top) is written: 5'→3' Template strand (bottom) is written: 3'→5' • Gene is transcribed from 5‘ end to the 3' end • Template strand of DNA is copied from the 3' end to the 5' end • Growth of RNA chain proceeds 5'→3' Orientation of a gene Transcription Complex Assembles at a Promoter • Consensus sequences are found upstream from transcription start sites • DNA-binding proteins bind to promoter sequences (prokaryotes and eukaryotes) and direct RNA pol to the promoter site • The s subunit of prokaryotic RNA polymerases is required for promoter recognition and formation of the complex E. coli promoter (1) TATA box (-10 bp upstream from transcription start site (rich in A/T bp) (2) -35 region (-35 bp upstream) from start site • A (sigma) subunit of RNA pol also required • Strong promoters match consensus sequence closely (operons transcribed efficiently) • Weak promoters match consensus sequences poorly (operons transcribed infrequently) • Subunit increases the affinity of the core polymerase for specific promoter sequences • Subunit also decreases the affinity of the core polymerase for nonpromoter regions • Core polymerase (no subunit) binds DNA nonspecifically RNA Polymerase Changes Conformation • Unwinding of DNA at the initiation site requires a conformational change • RNA pol (R) and promoter (P) shift from: (RPc) closed complex (DNA double stranded) (RPo) open complex (18 bp DNA unwound) (forms transcription bubble) Transcription Termination • Only certain regions of DNA are transcribed • Transcription complexes assemble at promoters and disassemble at the 3’ end of genes at specific termination sequences • Two types of termination sequences: (1) Unstable elongation complex (2) Rho-dependent termination Pause sites • Pause sites - regions of the gene where the rate of elongation slows down (10 to 100-fold) or stops temporarily • Transcription termination often occurs here • G-C- rich regions are more difficult to separate than A-T rich regions and may be pause sites • Pause is exaggerated when newly transcribed RNA can form a hairpin Rho-dependent termination sites • Rho () is a protein factor that triggers disassembly of the transcription complex at some pause sites • Rho binds to ssRNA chain, destabilizing the RNA-DNA hybrid and terminating transcription Transcription in Eukaryotes Eukaryotic RNA Polymerases • Three different RNA polymerases transcribe nuclear genes • Other RNA polymerases found in mitochondria and chloroplasts Eukaryotic Transcription Factors • Same reactions as prokaryotic transcription • More complicated assembly of machinery • Binding of RNA polymerase to promoters requires a number of initiation transcription factors (TFs) RNA polymerase II • Transcribes protein-encoding genes and some small RNA encoding genes • Protein-encoding RNA synthesized by RNA pol II is called mRNA precursor (or hnRNA) • Many promotors recognized by RNA pol II contain the TATA box (consensus sequence TATAAA) • General transcription factors interact directly with RNA pol II