Current LectureMCB 250 1st Edition Lecture 14Outline of Current Lecture 1. Transcription2. TerminationCurrent Lecture1. Transcription- RNA holoenzyme (Core + sigma) recognizes sequences in the DNA (promotor) bind to it and melt the DNA to form the open complex formation- Promoter: site in the DNA where RNA polymerase binds. Includes all binding sites for regulatory proteins- Promoters recognized by the primary sigma factor (sigma 70 in E. Coli), and line up all the promoters - Consensus sequence: common among all sequences and have a consensus sequence on non template strand -10 and -35 upstream- The promoter is recognized as double stranded DNA because the sigma factor recognizesthe sequence like any other sequence-specific binding protein by forming hydrogen bonds with the base pairs in the MAJOR GROOVE2. Termination: - 2 types- Rho-independent (Intrinsic) terminationo Depends on hairpin structure in the RNA recognized by RNA polymerase and transcription stops and RNA is releasedo Polymerase cruses down DNA and transcript is being made. There are signals in the DNA that is transcribed into the RNA that makes a hairpin loop followed by a row of U’s that causes transcription to terminateo DNA sequence is very GC rich and a palindromeo GC rich because it is relatively stableo Followed by a stretch of U’so Stem loop structure followed by a string of U’s. The stem is GC rich. Sequence of the stem is irrevelant, just that it is a stem loop. The U’s at the end are important though. These notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.o RNA is being made, RNA is coming out the back of the polymerase and will fall off, the stem talks to the polymerase to tell it to stop. The string of U’s= relatively weak interaction with DNA and is easy to be pulled out. o Once it happens, RNA pol will fall off and it will be done- Some fraction of the genes in E. Coli have a recognizable transcriptional terminator, but NOT ALL!!!!- Another mechanism terminates transcription: RHO- DEPENDENT- Rho: is a protein that binds to the RNA and causes the polymerase to stop transcribing. Half the terminators are Rho Dependent. RHO can also cause transcription termination ifthe ribosome stalls on the message.- Rho is a helicase that acts on RNA/DNA hybrid molecules, use ATP and strip RNA out of the back of the polymerase- mRNA are translated into protein. There are signals in the message that initiates the start of translation, ribosomes will bind and translates until stop codon. Prokaryotes translation and transcription are COUPLED. Before transcription is done, translation begins- Polymerase travels down DNA making RNA and as soon as the 5’ end comes out, ribosomes load and translate the message as RNA pol is transcribing the message- The ribosome is chasing the polymerase down the DNA.. There is data that suggests thatthe ribosome is pushing the polymerase down the DNA- Some point have a stop codon and the ribosome will stop. But the polymerase will keep going, but there is no Ribosome pushing it. There is naked RNA there. At some point at time, the ribosome will stop and the mRNA after that will be naked and nothing is covering it. - RHO comes in and will bind naked RNA. o RHO does have some sequence specificity. o RHO loads to the mRNA and uses ATP to travel 5’-3’ down the RNA and when it catches up the to polymerase, it will pull the mRNA out the back. o Helicaseo Don’t need intrinsic terminator at the end of the gene. Open reading frame, transcript it, when translation stops, polymerase keeps going but rho comes and stops transcription. Don’t need a real signal at the end because it will stop