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- Three types of RNA:o Messenger RNAs (mRNAs) – encode the amino acid sequence of one or more polypeptides specified by a gene or set of geneso Transfer RNAs (tRNAs) – read the information encoded in the mRNA and transfer the appropriate amino acid to a growing polypeptide chain during protein synthesiso Ribosomal RNAs (rRNAs) – constituents of ribosomes- Transcriptome – the sum of all the RNA molecules produced in a cell under a given set of conditions- Much of the genome of human and other mammals is transcribed into RNAo The products are primarily special-function RNAs26.1 DNA-Dependent Synthesis of RNA- Transcription does not require a primer- Only one DNA strand serves as a templateRNA Is Synthesized by RNA Polymerases- DNA-dependent RNA polymerase – an enzyme that can form an RNA polymerfrom ribonucleoside 5’-triphosphateso Requires ATP, GTP, UTP and CTPo Also requires Mg2+- RNA polymerase elongates an RNA strand by adding ribonucleotide units to the 3’-hydroxyl endo 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 replicationo Of less consequenceo Can remove mismatched bases by reversal of the RNA polymerase reactionRNA Synthesis Begins at Promoters- Promoters – specific sequences to which RNA polymerase binds to initiate transcription- The promoter region in E. coli extends from -70 to +30.- Consensus sequence – formed by certain nucleotides that are particularly common at each position- The UP element occurs between -40 and -60o Bound by the subunit of RNA polymerase- Pathway of initiation has two steps:o Binding of polymerase to promotero 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 degradationo Postsynthetic modificationso A specialized class of anti- proteinsTranscription 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 expressiono cAMP receptor protein (CRP) – a protein that activates transcription of genes coding for enzymes that metabolize sugars other than glucoseo Repressors – proteins that block the synthesis of RNA at specific genesSpecific 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 hairpinso 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 ruto Has an ATP-dependent RNA-DNA helicase activity that promotes translocation of the protein along the RNAo ATP is hydrolyzed in the termination processEukaryotic Cells Have Three Kinds of Nuclear RNA Polymerases- Eukaryotes have three RNA polymerases:o I – responsible for the synthesis of pre-ribosomal RNAo II – primary function is synthesis of mRNAs and some specialized RNAs Have a TATA box near base pair -30 Have an Inr sequence at +1o III – makes tRNAs, the 5S rRNA, and some other small specialized RNAsRNA Polymerase II Requires Many Other Protein Factors for Its Activity- Polymerase II has a long carboxyl-terminal tail consisting of many repeats of a consensus heptad amino acid sequence –YSPTSPS-- Transcription factors – other proteins required by RNA polymerase II- General transcription factors – highly conserved in all eukaryotes and required at every Pol II promoter- Several phases of transcription by Pol II:o Assemblyo Initiationo Elongationo Termination- Assembly of RNA Polymerase and Transcription Factors at a Promotero The formation of a closed complex begins when the TATA-binding protein binds to the TATA boxo TBP is bound by the transcription factor TFIIB, which also binds to DNA on either side of TBP Binding of TFIIA can stabilize the TFIIB-TBP complexo The TFIIB-TBP complex is bound by another complex of TFIIF and polymerase II TFIIF helps target polymerase II to its promoterso TFIIE and TFIIH bind to create the closed complex TFIIH has DNA helicase activity- RNA Strand Initiation and Promoter Clearanceo 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 Releaseo TFIIF remains associated with polymerase II throughout elongationo Elongation factors enhance the polymerase activity- Regulation of RNA Polymerase II Activity- Diverse Functions of TFIIHo 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 complexo Xeroderma pigmentosum and Cockayne’s syndrome are caused by genetic loss of certain TFIIH subunitsDNA-Dependent RNA Polymerase Undergoes Selective Inhibition- Actinomycin D – an antibiotic that inhibits elongation of RNA strands by RNA polymerase in both bacteria and eukaryoteso Intercalates into the double-helical DNA between G-C pairs, deformingthe DNAo Used to identify cell processes that depend on RNA synthesis- Acridine – inhibits RNA synthesis in a similar fashion to actinomycin D- Rifampicin – inhibits bacterial RNA synthesis by binding to the subunit of bacterial RNA polymerases, preventing the promoter clearance step of transcriptiono Sometimes used as
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