Lecture 9 RNA Structure Synthesis and Processing 1 Compare the structure of DNA and RNA understanding the difference between the constituent bases sugars nucleosides and nucleotides DNA Deoxyribonucleic acid Double stranded sugar phospore Bases are A C G and T The sugar is deoxyribose DNA can self replicate and is read by polymerase 3 5 Types nuclear and mtDNA Usually only one strand of DNA is used as a template RNA Ribonucleic acid Usually single stranded Bases are A C G and U The sugar is Ribose Stores info about protein structure Read by ribosomes and is formed in 5 3 There are many types of RNA such as transfer ribosome and messenger RNAs RNA can be initiated de novo with no primer and it is catalyzed by RNA polymerases The precursors to RNA are ribonucleotide phosphates like ATP GTP CTP and UTP RNA is highly selective 2 Describe the subunit structure of RNA polymerase from E coli and assign functions to the individual subunits In E Coli two DNA sequences act as promoters for many genes These are the Pribnow box and the 35 sequence Transcription RNA synthesis is a process catalyzed by RNA polymerase RNA polymerase must have a template activated precursors and divalent meta ions RNA polymerase has three subunits One subunit of RNA polymerase is the Sigma subunit This is where the initiation step occurs The sigma subunit of RNA polymerase recognizes promoter sites which are specific DNA sequences that direct RNA polymerase to the proper initiation site The sigma subunit also assists the enzyme in identifying the promoter And once the promoter is located and RNA synthesis starts the sigma subunit dissociates from the enzyme to go help another polymerase initiation somewhere else Another subunit is the alpha subunit There are two of these This is part of the catalytic domain that elongates the RNA with 5 3 RNA polymerase activity In this region during elongation RNA polymerase continues binding bond formation and translocation cycles In RnA no primer is required for elongation and this process has helicase activity DNA topoisomerases I and II relax supercoils Ribonucleoside triphosphate releases pyrophosphate forming phosphodiester bonds The Beta subunit recognizes the termination signals There are two beta subunits This is where the termination step occurs The ends of genes are recognized by RNA polymerase complexes 3 List the salient sequence elements of eukaryotic promoters Contrast the nucleotide sequences and locations of the TATA box of eukaryotes and the 10 sequence of prokaryotes The steps to RNA synthesis are initiation elongation and termination The promoter region is the DNA sequence that allows and directs RNA polymerase to build off of it Prokaryotic E coli has two DNA sequences The Pribnow Box 10 and the 35 sequences Eukaryotic TATA CAAT and GC boxes are cis acting elements in eukaryotic promoters that are recognized by proteins other than RNA polymerase The caAT box is located between 40 and 150 The TATA box is usually centered at roughly 25 TATA boxes are similar to the bacterial 10 sequences TATAAT Pribnow Box in prokaryotes but are farther from the start site The AT rich sites have bonds that are easier to break and thus these segments of DNA can be readily split 4 Understand the dependent and independent transcription termination Rho independent Intrinsic termination In this process the RNA transcript forms a Termination can be both p dependant and independent p rho stable hairpin turn or a stem loop RNA transcripts also contain a string of U s Rho independent termination is dependent on the sequence of DNA Rho dependant protein dependent In this process the rho factor dissociates from a polymerase and binds to a C rich region that contains helicase activity from ATP hydrolysis This unwinds the 3 end of the transcripts from the template which displaces the DNA template strand 5 Describe the mechanisms of inhibition of transcription by rifampicin actinomycin D doxorubicin and alpha amanitin Rifampicin inhibits initiation or elongation It can block transcription initiation by binding to the polymerase on DNA It can also inhibit elongation by binding to DNA bound polymerase Actinomycin D This compound intercalates between the bases of the DNA double helix which prevents DNA from being used as a template This prevents RNA elongation Doxorubicin A intercalating agent that distorts the DNA double helix structure This is used to treat Hodgkin s lymphoma Alpha amanitin This comes from a poisonous mushroom that has compounds that target eukaryotic RNA polymerase II RNA polymerase II helps with mRNA synthesis so alpha amanitin is a selective inhibitor of RnA synthesis 6 Understand the structure of a typical human protein coding gene including upstream sequences promoter 5 UTR transcription start site coding sequences introns 3 UTR polyadenylation site In DNA 5 Enhancer Promoter transcription start site Exon Introns Poly A signal sequence Transcription termination region 3 Transcription start site This is where transcription begins Promoter a region in the middle that allows Polymerase to start building Enhancer a digital control element poly A signal sequence Transcription termination region the place where transcription termination occurs Exon coding sections of RNA proteins Intron non coding sections of RNA proteins UTR untranslated region Mature RNA can be used to synthesize proteins while pre RNA cannot until it is matured through RNA Processing 5 5 Cap 5 UTR Start codon code stop codon 3 UTR Poly A tail 7 What is a ribozyme Can you give an example of the reactions carried by ribozymes Ribozymes are catalytic RNAs that function as enzymes They are able to cleave and ligate RNA Natural ribozymes occur within self splicing introns and RNA encoded parasites Reaction Examples RNAse P cleaves 5 end of pre tRNAs catalytic activity lies entirely w i RNA Phosphodiester link cleavage catalyze cleavage and ligation ANGIOZYME Ribozyme designed to inhibit Vascular Endothelial Growth Factor HEPTAZYME Ribozyme targets highly conserved sequences of Hepatitis C part VEGF virus 8 Describe the post transcriptional processing of eukaryotic mRNA including capping splicing and polyadenylation Capping a 7 methyl guanosine cap is added to the 5 end of the primary transcript by a 5 5 phosphate linkage This permits the initiation of translation and stabilizes the mRNA Capping protects from exonucleases which degrade RNA Splicing splicing enzymes are small nuclear ribonucleoprotein