Chapter 7From DNA to protein: How cellsread the genomePart 1: TranscriptionThis is key to• understanding why different cells in a plant or animal have different properties• understanding how levels of specific proteins are regulated• understanding how many signals and drugs affect cells• increasing yields of a protein in biotechnology• decreasing levels of an enzyme to for metabolic engineering• thinking of ways to turn off a ‘bad’ gene in medicineObjectiveUnderstand the basic mechanism of transcription ineukaryotes and prokaryotes•Describe the substrates used by RNA polymerase•Compare these to the substrates used by DNA polymerase•Explain which strand has the key information used by RNA polymerase, and defend your answer•Given a promoter site and a DNA sequence, write the resulting RNA sequence•Describe the different types of RNA•Describe a promoter•Explain why only some regions of DNA are transcribed to RNA, and explain how the strand in this region is chosen•Compare prokaryotic and eukaryotic transcription•Compare and draw prokaryotic and eukaryotic gene structure•Describe eukaryotic RNA processing•Explain the difference between introns and exons•Sketch a splicing reactionBe#able#to:Transcription provides amplification of genetic informationGenes can be transcribedwith very different efficienciesTranscription produces RNAcomplementary to one strand of DNA5’-AGGTCCACG-3’3’-TCCAGGTGC-5’DNATemplate strandTranscription (5’ to 3’)5’-AGGUCCACG-3’RNATranscriptionA small portion of DNA helix must be opened and unwoundto expose the bases on each strandRNA is synthesized by RNA polymerase• Nucleotides added to 3’ end of RNA strand (5’ to 3’)• RNA sequence is dependent on complementary basepairing (A-U, G-C)• No primer is neededThe complementary RNA strand is released from the DNAtemplate strand almost immediately. This allows anotherRNA polymerase molecule to initiate even before the first iscompeted.RNA polymerase error rate is 1 in 104. Compare to DNA polTranscription of DNA by RNA polymeraseGenes can be simultaneously transcribedby many RNA polymerase moleculesAmplification of genetic informationDirection?What controls where RNA polymeraseinitiates and terminates transcription?Promoter: DNA sequence that is recognized by RNApolymerase as a start point.Chain elongation occurs until RNA polymerase reaches aterminator site, at which RNA is released and the RNApolymerase dissociates from the DNABoth strands can serve as a template for RNA polymerase07_09_1_bacterial gene.jpgTranscription in bacteria07_09_2_bacterial gene.jpgPromoters are asymmetrical so RNA polymerasetranscribes in only one directionTranscription in bacteriaTranscription in eukaryotes differs from prokaryotes1. RNA polymerase• Prokaryotes have a single type• Eukaryotes have three typesRNA pol I: most rRNA genesRNA pol II: protein-encoding genes (makes mRNA)RNA pol III: tRNA, 5S rRNA, small structural RNA genes2. Initiation• Prokaryotic RNA pol can initiate without helper proteins• Eukaryotic RNA pols require general transcription factors3. Transcript processing• Prokaryotic transcripts are generally NOT processed• Eukaryotic mRNAs are processedTATA-binding protein (TBP) isa subunit of TFIIDAssembly of transcriptioninitiation complexPhosphorylation of RNA Pol IIby TFIIH, transcriptionproceedsTranscription initiationRNA pol IIPhosphorylation ofRNA Pol II by TFIIHalso allows RNAprocessing proteinsto assemble on itstailStopping transcription is badα-amanitin is made by the ‘death cap’mushroomTranscription initiation by RNA polymerase IIStudy guideKey steps:• General transcription factor TFIID binds to TATA boxTATA box is a conserved sequence found in nearly allRNA pol II-transcribed promoters and is located ~25base pairs upstream of transcription start sites• Assembly of transcription initiation complex on promoterIncludes other general factors and RNA Pol II• Disengage RNA pol II from complex to begin transcriptionPhosphorylation of RNA pol II tail by TFIIH (kinase)• General factors release from DNA once transcription beginsRNA processing in eukaryotic cellsTranscription takes place in the nucleus and translation(protein synthesis) takes place in the cytosolEukaryotic RNA must be transported from nucleus to cytosolBefore leaving the nucleus, mRNA must be processed• RNA capping (modification of 5’ end, 7-methylG)• Polyadenylation (modification of 3’ end, polyA tail)• Splicing (removal of introns)Prokaryotic RNA is generally not processedGene organization differs between eukaryotes and prokaryotes07_12_capping.jpgpolyA tail(polyA polymerase)Capping and polyadenylationincreases stability of eukaryoticmRNA moleculesPolycistronic gene organizationMonocistronicIntrons are removed by RNA splicingAn entire gene is transcribed (exons plus introns) butremoval of the introns begins immediately after cappingoccurs. Capping and splicing both occur while RNApolymerase continues to transcribe DNA.RNA splicing is performed largely by catalytic RNAmolecules (small nuclear RNAs, snRNAs) with helpfrom a few proteins (snRNPs). SpliceosomeThe splicing RNA molecules recognize intron-exonboundaries or junctions (sequence-specific recognition)and cut out introns as a lariat structure07_16_RNA_chain .jpgThe sequence of only a few small partsof an intron are critical for splicing5’ splice junction 3’ splice junctionAlternative splicing provides a mechanismfor functional diversityTissue-specific splicing variantsSplicing errors usually leadto nonfunctional proteinsMature mRNAs are selectively exported from the nucleus Only mature mRNA is exported - no introns, broken strandsor incorrectly spliced variants. These are distinguished bythe binding of specific proteins associated with each step ofRNA processing:• polyA-binding proteins• Cap-binding complex• Nuclear transport receptor07_20_Pro_v_Eucar.jpgProkaryotes andeukaryotes handletheir transcriptsdifferentlyChapter 7From DNA to protein: How cellsread the genomePart 2: TranslationThis is key to• understanding, and predicting the effects of, many mutations• genetic engineering - making a precise modification to a protein• understanding the mechanism of action of many antibioticsObjectiveUnderstand translation and the basic mechanism of geneexpression•Explain codon redundancy•Predict the proteins encoded by a piece of DNA or RNA•Write a DNA or RNA sequence that would encode