BSCI222 – Lecture 4 (9/12/13)- RNA Transcription (Ch. 13 and 14)o U instead of T, hydroxyl group on the 2’ Carbon of the sugar, whereas DNA has aHydrogen there. RNA is more reactive than DNA.o RNA can take on many shapes, because the single strands folds up on itself to satisfy its Hydrogen requirements.o DNA is thought to have become the genetic material through evolution because it’s much more stable than RNA (because RNA is single stranded and its chemical structure).o DNA’s secondary structure is usually the Beta-helix; RNA’s primary structure is linear, secondary is folding (with base pairing, and some twisting). (stems and loops)- Many kinds of RNA:o Of course mRNA, rRNA (ribosomal, structural and functional components of the ribosome), and tRNA, but also many tiny ones that we’re still learning their functions.- RNA is transcribed from the DNA (NOT translated; transcription is a copy of base pairs, translation is turning it into protein language instead of nucleic acid language). - RNA is always synthesized 5’ to 3’ too, and antiparallel to the template strand. - Always report DNA 5’ to 3’. But that’s not the strand that is transcribed into RNA; RNA is transcribed from the template strand, the complimentary strand (3’ to 5’). (RNA is made from the bottom strand, the 3’ to 5’, therefore the RNA is made 5’ to 3’) Either strand of the DNA could encode the gene.o Promoter (“upstream”) – RNA coding region – Terminator (“downstream”) (stop the RNA from transcribing, so the polymerase doesn’t go all the way around to the end of the chromosome and make an enormous molecule. Then there’d be no control over the expression of genes, they’d all be transcribed).- RNA transcription/synthesis:o Helix has to be opened up to give us access to the bases and Hydrogen bonds, in order to specify which nucleotides should be synthesized.o Don’t need a primer, RNA polymerase can start making RNA on a single nucleotide strand.o Transcription bubble moves along the helix, closes up behind the polymerase.o PROKARYOTIC:To know a sequence is functional, have to find sequences that are always found upstream or downstream of a gene. Consensus sequences: compare the actual sequences, find common patterns (almost all have a T first, consensus sequence starts with T. Equalproportion of T or C, both pyrimidines, next letter is Y. N = no pattern.) Typically find a consensus sequence 10 sequences before the transcription start site (called the minus ten box, TATAAT) and 35 sequences before (called the minus thirty five box, TTGACA). If every gene had exactly the same sequences upstream, no control over gene expression. Variation (closer to consensus sequence or farther away from it; it is perfect and the strongest sequence for a protein to bind to, fitting the protein when binding perfectly) will lower or raise protein binding, strength of gene expression. A subunit of the RNA polymerase binds to the minus ten box and minus thirty five box, called the Sigma factor (the other subunits bind anywhere, no specificity). The Sigma factor is what gives the polymerase specificity so it only binds to promoters and increases the rate of transcription by helping it bind more tightly. Essentially, the Sigma factor controls which genes will be expressed. Core RNA polymerase + Sigma bind to promoter, creating a closed complex (of six different proteins). Holds all those subunits tightly to the promoter. The holoenzyme (RNA polymerase) binds to the promoter and unwinds the helix (“open promoter complex”), beginning the process. o RNA polymerase has to be stopped once it gets to the end of the gene. Two mechanisms: Rho-protein independent mechanism: A particular sequence at the end of the gene, of repeats of A-U, which folds up into a hairpin loop, causing theRNA polymerase to pause (clogs it up). Stall the polymerase and then the base pairs that are A-U (only 2 hydrogen bonds, instead of 3) are weak, big chance that thermal motion will cause the hairpin loop to completely fall off. Rho-protein dependent: Still a stem-loop that makes RNA polymerase stall, but then Rho-protein binds to the transcriptor and starts chasing the polymerase (catches up when it pauses), then has a gyrase activity that separates that transcript from the helix. Always key to slow down the polymerase long enough for the transcript tobe disassociated.o DNA being transcribed into RNA looks like a Christmas tree (gene start is at the top, where the RNA strands are short. Trunk is DNA. As polymerase moves downthe trunk, RNA “branches” get longer, more has been transcribed). Little ornament balls on the end of the branches might be ribosomes, meaning prokaryotic (only place you can have simultaneous transcription and translation). Each branch has individual polymerase.o EUKARYOTES: RNA polymerases are specialized. Pol II is doing most of the messenger RNAs and some of the smaller ones. Pol III does the tRNAs and some of the smaller ones. Pol I only does the large ribosomal RNAs. As eukaryotic genome got more complex, had to figure out how to regulate transcription on a much larger scale. Pol II is huge, DNA goes through it, RNA exits out as a single strand. Consensus sequences (not the same as prokaryotic): -25 box (called TATAbox, TATAAA), +1 (downstream of start site) initiator element, +30 box (downstream core promoter element).- Prokaryotes’ specificity is more consensus sequences and binding strengths. Eukaryotes: have core promoter but also much larger regulatory promoter site upstream of the start site. Will find recognizable promoter sites, in different arrangements and different numbers in front of different genes. Will affect how muchand when the gene is expressed.- Their exact position doesn’t matter. You’d think the binding site would have to be in an exact place, but it’s actually very flexible, get essentially the same gene expression. It’s the Presence and number of these boxes that matters.  Prokaryotic transcription complex (core + sigma); eukaryotes’ is much bigger.- Have TBP (“TATA binding protein”) bind to TATA box, transcription factor 2D is recruited, other proteins begin to assemble into the core transcription complex (or basal transcriptional apparatus). Giant conglomeration of proteins, all interacting with each other to localize the polymerase to the exact spot that the transcription should start, increasing its