Which strand to transcribe determine by orientation of promoter sequence Transcription Step 1: Initiation ~ RNAP holoenzyme binds promoter region s subunit binds – 35 & 10 region (recognizes sequences) ~ DNA strands unwind beginning at – 10 region to form transcription bubble providing rRNP access to template strand ~ Formation of phosphodiester bonds generating mRNA s factor release after ~ 10 nucleotides synthesized RNAP core proceeds to elongate mRNA Abortive synthesis of short transcripts (2 – 9 nucleotides) precedes productive synthesis Transcription Step 2: Elongation ~ Extension of RNA chain by polymerase core (a 2 bb’) ~ Polymerization occurs within transcription bubble (18 bp) ~ Adds 40 ribonucleotides / sec as move along DNA ~ Core enzyme unwinds DNA ahead / rewinds behind polymerization site ~ Growing RNA chain displaced as made (DNA – RNA hybrid ~ 3 bp long) Transcription Step 3: Termination ~ Chain termination occurs when RNAP encounters termination signal ~ RNA polymerase / DNA complex dissociates ~ RNA transcript released ~ Two types of termination signals: Rhoindependent terminators Rhodependent terminators – requires rho (r) protein binding Both depending upon formation of hairpin structure upstream of termination site ~ Rhoindependent terminators
Contain G:C rich inverted repeats followed by ~ 6 As on template strand G:C rich inverted repeats form singlestranded RNA hairpin structure Hairpin structure impedes further movement of RNAP along DNA Paused RNAP causes weakly bonded RNA/DNA hybrid to separate Release of RNA transcript causes dissociation of RNAP ~ Hairpins cause issues in continuation of DNA growth ~ Rhodependent terminators Lack 6 nucleotide AT stretch, only some contain hairpin structure Rho is hexameric, has RNA binding & ATPase activity Rho binds rhobinding site rut (rho utilization) near 3’ end of transcript Rho moves along RNA until encounters RNAP Rho does one of two things: - Pulls RNA out of bubble - “Pushes” RNAP forward along DNA, thus pulling RNA out of complex Transcription & translation coupled in prokaryotes ~ Several polymerases can transcribe gene simultaneously ~ RNA transcript translated into protein concurrently (ie. protein made as synthesis occurring) ~ Degradation of transcript can occur before transcript is complete Lecture 7: Transcription in Eukaryotes (chapter 11, pg. 267 – 274) ~ 3 different polymerases: RNA Polymerase I, II, & III ~ Basal transcription initiation complex & transcriptional activators ~ PremRNA modification: capping, tailing, splicing, editing Transcription in Prokaryotes: ~ Only 1 RNA polymerase ~ mRNAs multigenic ~ Gene expression controlled by regulation rate of transcription (mRNA halflife usually < 5 min) ~ Regulation of gene expression: Promoter strength Enhancer binding
Repressor binding Three main* eukaryotic RNA polymerases ~ Types of RNA: mRNA – code for protein tRNA – adaptor between mRNA & amino acids in protein synthesis rRNA – component of ribosomes (28S, 18S, 5.8S, 5S)
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- Fall '13
- DNA, DNA Transcription & RNA Processing