Chapter 26 RNA Metabolism Transcription when an enzyme system converts the genetic information in a segment of double stranded DNA into an RNA strand with a base sequence complementary to one of the DNA strands o Messenger RNAs mRNAs encode the amino acid sequence of one Three types of RNA or more polypeptides specified by a gene or set of genes and transfer the appropriate amino acid to a growing polypeptide chain during protein synthesis rRNAs constituents of ribosomes tRNAs read the information encoded in the mRNA o Transfer RNAs o Ribosomal RNAs the sum of all the RNA molecules produced in a cell under a Transcriptome given set of conditions Much of the genome of human and other mammals is transcribed into RNA o The products are primarily special function RNAs 26 1 DNA Dependent Synthesis of RNA Transcription does not require a primer Only one DNA strand serves as a template RNA Is Synthesized by RNA Polymerases DNA dependent RNA polymerase from ribonucleoside 5 triphosphates o Requires ATP GTP UTP and CTP o Also requires Mg2 an enzyme that can form an RNA polymer RNA polymerase elongates an RNA strand by adding ribonucleotide units to the 3 hydroxyl end o The 3 hydroxyl group acts as a nucleophile attacking the phosphate of the incoming ribonucleoside triphosphate and releasing pyrophosphate Initiation occurs when RNA polymerase binds at specific DNA sequences called promoters DNA dependent RNA polymerase has five core subunits 2 and a sixth subunit that binds transiently to the core and directs the enzyme to binding sites on the DNA The error rate for transcription is higher than that for chromosomal DNA replication reaction o Of less consequence o Can remove mismatched bases by reversal of the RNA polymerase RNA Synthesis Begins at Promoters Promoters transcription specific sequences to which RNA polymerase binds to initiate The promoter region in E coli extends from 70 to 30 Consensus sequence common at each position formed by certain nucleotides that are particularly The UP element occurs between 40 and 60 o Bound by the subunit of RNA polymerase Pathway of initiation has two steps o Binding of polymerase to promoter o Initiation leading to a conformational change that converts the complex to the elongation form What sets of genes are expressed is determined by which subunits are available which is determined by several factors o Regulated rates of synthesis and degradation o Postsynthetic modifications o A specialized class of anti proteins Transcription Is Regulated at Several Levels Much of the regulation is directed at the polymerase binding and transcription initiation Differences in promoter sequences are one of several levels of control The binding of proteins to sequences both near to and distant from the promoter can also affect levels of gene expression CRP a protein that activates transcription o cAMP receptor protein of genes coding for enzymes that metabolize sugars other than glucose o Repressors proteins that block the synthesis of RNA at specific genes Specific Sequences Signal Termination of RNA Synthesis RNA synthesis is processive Most independent terminators have two distinguishing features o A region that produces an RNA transcript with self complementary sequences permitting the formation of hairpins o A highly conserved string of three A residues in the template strand The dependent terminators lack the sequence of repeated A residues but usually include a CA rich sequence called a rut o Has an ATP dependent RNA DNA helicase activity that promotes translocation of the protein along the RNA o ATP is hydrolyzed in the termination process Eukaryotic Cells Have Three Kinds of Nuclear RNA Polymerases Eukaryotes have three RNA polymerases o I responsible for the synthesis of pre ribosomal RNA o II primary function is synthesis of mRNAs and some specialized RNAs Have a TATA box near base pair 30 Have an Inr sequence at 1 o III makes tRNAs the 5S rRNA and some other small specialized RNAs RNA Polymerase II Requires Many Other Protein Factors for Its Activity Transcription factors General transcription factors Polymerase II has a long carboxyl terminal tail consisting of many repeats of a consensus heptad amino acid sequence YSPTSPS required at every Pol II promoter Several phases of transcription by Pol II other proteins required by RNA polymerase II highly conserved in all eukaryotes and o Assembly o Initiation o Elongation o Termination Assembly of RNA Polymerase and Transcription Factors at a Promoter o The formation of a closed complex begins when the TATA binding o TBP is bound by the transcription factor TFIIB which also binds to protein binds to the TATA box DNA on either side of TBP Binding of TFIIA can stabilize the TFIIB TBP complex o The TFIIB TBP complex is bound by another complex of TFIIF and polymerase II TFIIF helps target polymerase II to its promoters o TFIIE and TFIIH bind to create the closed complex TFIIH has DNA helicase activity RNA Strand Initiation and Promoter Clearance o Kinase activity in one of the subunits of TFIIH phosphorylates polymerase II at many places in the CTD causing a conformational change that initiates transcription Elongation Termination and Release o TFIIF remains associated with polymerase II throughout elongation o Elongation factors enhance the polymerase activity Regulation of RNA Polymerase II Activity Diverse Functions of TFIIH o TFIIH participates in formation of the closed complex during assembly of the transcription complex Some of its subunits are also essential components of the separate nucleotide excision repair complex o Xeroderma pigmentosum and Cockayne s syndrome are caused by genetic loss of certain TFIIH subunits DNA Dependent RNA Polymerase Undergoes Selective Inhibition Actinomycin D polymerase in both bacteria and eukaryotes an antibiotic that inhibits elongation of RNA strands by RNA o Intercalates into the double helical DNA between G C pairs deforming the DNA o Used to identify cell processes that depend on RNA synthesis Acridine Rifampicin inhibits RNA synthesis in a similar fashion to actinomycin D bacterial RNA polymerases preventing the promoter clearance step of transcription inhibits bacterial RNA synthesis by binding to the subunit of o Sometimes used as an antibiotic Amanitin a compound produced by a mushroom that disrupts mRNA formation in animal cells by blocking polymerase II and polymerase III Primary transcript 26 2 RNA Processing Ribozymes Noncoding segments are introns
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