University:
Glendale Community College
Course:
BIO160 | Introduction to Human Anatomy and Physiology
Academic year:

2020
Views:

335

Pages:

14
Author:

helkoktcj

Section 18.2: Transcription Transcription – creation of RNA from DNA sequences §RNA polymerase – enzyme catalyzing the addition of ribonucleotides in a 5′à3′ direction §All 4 ribonucleoside triphosphates required, Mg2+ §Primer is not needed, DNA template is required §Only 1 strand of helix is used §Nontemplate strand (+) - same base sequence as the RNA, except transcript has uracil for thymine §Template strand (-) - antiparallel to the new RNA strand §Contains initiation & termination signals §Enzyme moves in 3’-to-5’ direction §RNA created in 5’ à 3’ Figure 18.31 DNA Coding Strand Section 18.2: Transcription Figure 18.30 E. coli RNA Polymerase Transcription in Prokaryotes §RNA polymerase in E. coli catalyzes the synthesis of all RNA classes §Core enzyme (a2,b and b′) catalyzes RNA synthesis §Holoenzyme – complete enzyme (a2wbb’s) §s subunit promotes core assembly and s-factor functions in transcription initiation §Promoters – RNA polymerase binds to DNA at start of transcription Section 18.2: Transcription RNA Polymerase slides down DNA 3’ -> 5’ to promoter sequence §Upstream of start of transcription §3 components – 2 consensus sequences, 3rd TSS §Transcription start site (TSS) -position +1 §Consensus sequences at -10 region (Pribnow box) and -35 region §Core promoter - -35 element to TSS §Chain initiation – RNA pol recognizes -35 region §Closed complex – forms when promoter binds Figure 18.32 Typical E. coli Transcription Unit §Open complex - break hydrogen bonds; unwind a short segment of DNA at Pribnow box §RNAP catalyzes the addition of first nucleoside triphosphate §10 nucleotides synthesized – RNAP clears promoter region §s factor is released, ready for elongation Section 18.2: Transcription §Transcription bubble - unwound DNA of 12–14 bp §Contains RNA-DNA hybrid §Template strand enters active site through channel §Active site lies between b/b’ subunits §Nontemplate strand loops away from active site into its own channel §Both strands emerge, re-forming a double helix Section 18.2: Transcription Figure 18.31 Transcription in E. coli Growing RNA chain exits through a channel ü Positive supercoils ahead of transcription; negative supercoils behind ü Topoisomerases relieve the supercoils, advance bubble ü No relax – RNA chain wraps around helix every 10bp §Termination signal - ends transcription Section 18.2: Transcription Figure 18.34 Intrinsic Termination §Intrinsic termination – controlled by termination sites §2 inverted repeats spaced by few other bases §Sequences of complementary bases, loop back on themselves §DNA encodes a series of uracils §Forms a stable hairpin causing RNA polymerase to slow or stop §Series of A-U base pairs between template strand & RNA §RNA transcript is released due to weak base-pair interactions Section 18.2: Transcription rho-dependent termination – aid of ATP-dependent helicase rho factor §Rho binds to a specific recognition sequence on the nascent RNA chain, upstream from termination site §Chases polymerase; when hairpin loop forms, stalls allowing rho to catch up Figure 18.35 Rho-Dependent Termination §Unwinds the RNA-DNA helix to release the transcript Section 18.2: Transcription Transcription in Eukaryotes §Polymerases are similar in structure and function – 1 vs 3 §Initiation factors are distantly related, but perform similar functions §Regulatory mechanisms differ significantly in both organisms §One major difference is the limited access to DNA of the transcription machinery §Chromatin least partially condensed §Transcription premissive - histone tails modified by histone acetyl transferases (HATs) §Chromatin remodeling complexes – weakens histone-DNA contacts §SWI,SNF, and NURF Section 18.2: Transcription §RNA Polymerase Activity §RNA polymerase I (RNAPI) transcribes larger rRNA (28S, 18S, and 5.8S) in the nucleolus §RNA polymerase II (RNAPII) produces the precursors of mRNA, miRNAs and most snRNA §RNA polymerase III (RNAPIII) is responsible for transcribing the precursors for tRNA, 55 rRNA, U6 snRNA, and the snoRNAs §Requires various transcription factors bound to the promoter to initiate transcription Section 18.2: Transcription Eukaryotic promoters- Pol II core promoter can be focused or dispersed §Focused - transcription start site (TSS) and core promoter elements (CPE) §TATA box - consensus sequence, TATAA §TFIID - TATA-binding protein (TBP) binds to TATA box. §Other core elements include the Inr (initiator), BRE (B recognition element), and DPE (downstream promoter element) §Dispersed genes -multiple TSSs, distributed over a broad region of 50-100 basepairs §Typically occur within CG islands §Facilitate nucleosome destabilization Section 18.2: Transcription RNA polymerase II (RNAP II) §12 subunits in humans §RBP1, largest subunit, forms part of the enzyme’s active site; binds DNA §Contains C-terminal domain (CTD) §Unphosphorylated allows RNAPII to bind promoters §RNAP II machinery - set of five transcription factors and a 20protein mediator complex §General transcription factors (GTFs): TFIIB, TFIID, TFIIE, TFIIF, and TFIIH are the minimum required for accurate transcription §Facilitate promoter recognition, preinitiation complex formation, and ATP-dependent DNA unwinding Section 18.2: Transcription §PIC assembly - binding of TBP subunit of TFIID to the TATA box. §Other GTFs: TFIIA, TFIIB, TFIIF, mediator §TFIIH acts as an ATP-dependent helicase §Promoter clearance occurs after 23 nt §Elongation - promoter clearance; mediator dissociation §Termination Sequence - poly(A) sequence, (5′-AAUAAA-3′) §Poly(A) tail- (100–200 adenylate residues) to end of the transcript Figure 18.44 Preinitiation Complex Formation at a TATA Box Section 18.2: Transcription Figure 18.45 The Methylated Cap of Eukaryotic mRNA §RNA Processing- enzymatic modification §Trimming -removal of leader (5’) and trailer (3’) sequences §capping – protects 5’ end from nucleases §Synthesized when the transcript is about 30 nt long Section 18.2: Transcription RNA splicing - removal of introns from an RNA transcript §Spliceosome – removes introns; links exons together forming a functional product §Splicing reaction 1. A 2′-OH of an adenosine nucleotide within intron attacks a phosphate in 5′ splice site, forming a lariat 2. Lariat is cleaved; two exons joined when 3′-OH of the upstream exon attacks a phosphate adjacent to the lariat §5′ splice site is the donor site and the 3′ splice site is the acceptor site §Four active spliceosomes form with each pre-mRNA to form a supraspliceosome Figure 18.47 RNA Splicing