BioMG 3320 1st Edition Lecture 7Outline of Past LectureI. Chemistry of RNAII. RNA as Genetic MaterialIII. RNA Structures Outline of Current LectureI. Basics of TranscriptionII. Transcription Cycle Current LectureI. Basics of Transcription- Transcription consists of unwinding DNA and using a single strand as a template to guide the synthesis of RNA- RNA polymerase adds ribonucleotides to the DNA strand, moving in the 5’ to 3’ direction- The RNA transcript is identical to the DNA nontemplate strand, with the exception of U nucleotides instead of T- Chemistry of transcription: incoming nucleotide pair is attacked at the alpha phosphate by the 3’ hydroxyl of the growing RNA chain- RNA polymerase is the protein machinery that makes RNA. It is an efficient enzyme-It winds double helix in front of it and rewinds it after it is finished 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.-Core enzyme consists of two alpha subunits, beta and beta’ subunits, and one omega subunit-Holoenzyme= core + a sigma subunit -Alpha: assembly; interacts with regulatory factors; catalysis-Sigma: promoter recognition-Beta: polymerase activity-Beta’: DNA binding-Omega: unknown function- Bacterial RNA polymerase: beta, beta’, alpha, alpha, omega- Yeast RNA polymerase is RNA polymerase II - E. coli has 7 different sigma factors so there is a potential for 7 different holoenzymes that would recognize different promoters- Phage DNA consists of genes recognized by housekeeping E.coli. It is transcribed and translated to make phage proteins with new sigma factors that have a high affinity for core enzyme. It displaces the core enzyme and makes RNA polymerase that recognizes a different promoter. Repeats. Result is 3 different sigma factors to achieve the lifecycle of the phage- Promoters contain a UP element, which makes a stronger promoter, a -35 and -10 regionand +1 region as the RNA start site. Sigma binds at the -10 region. Polymerase contacts with promoter elementsII. Transcription Cycle- Transcription consists of initiation, elongation and termination- Initiation consists of promoter recognition, transition from closed to open state, and promoter escape- Elongation consists of RNA synthesis, template unwinding and rewinding- Termination is transcript release- Initiation: -Promoter recognition: polymerase does not need a primer-Closed state: DNA still double stranded-Open state: unwind DNA-Promoter escape and beginning of synthesis -RNA polymerase falls off a few times before it stays on and makes RNA; makes short abortive chains and then gets back on. This is its way or regulation- Initiation Mechanisms:-Transient excursions: RNA polymerase falls off and then moves backwards on DNA-Inchworming: Polymerase has two parts linked together. Part of polymerase stays on position on promoter and other part with catalysis center moves-Scrunching: Polymerase and DNA in same position. Early nucleotides of DNA scrunched out. Reform to make another RNA if RNA polymerase falls off. This is most likely method- Elongation: sigma dissociates and proteins join, facilitating elongation- Correcting Errors:-Pyrophosphorolytic editing: same active site as polymerization; backward reaction- subtract nucleotide (there is a wrong nucleotide, go backwards and take it off)-Hydrolytic editing: enzyme backtracks; cleaves RNA containing errors- Termination ƿ-independent: sequence forms hairpin close to transcription causing polymerase to fall off DNA- Termination ƿ-dependent: ATP-dependent helicase recognizes rut elements (C-rich sequences) and ƿ binds nascent transcript and migrates toward polymerase; interactionsbetween RNAP and ƿ result in termination - Detecting specific RNA species through northern blotting:-RNA separation by gel electrophoresis requires denaturing gels since they would migrate due to size and shape. If denature, make same shape so they only separate by size-RNA blot on nitrocellulose-Detect probe, revealing RNA of