生物化学讲义Chapter 26 (complete) RNA Metabolism.ppt
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1、Chapter 26 RNA Metabolism,1. How is RNA synthesized using DNA templates (transcription)? 2. How is newly synthesized primary RNA transcripts further processed to make functional RNA molecules? 3. How is RNA and DNA synthesized using RNA as template (reverse transcription); 4.What is the evolutionary
2、 implication of the structural and functional complexity of RNA molecules?,1. RNA molecules have great structural and functional diversity,With structures comparable to proteins in complexity and uniqueness. Function as messengers between DNA and polypeptides (mRNA), adapters (tRNA) to match a speci
3、fic amino acid with its specific genetic code carried on mRNA, and the structural and catalytic components of the protein-synthesizing ribosomes (rRNA). Stores genetic information in RNA viruses. Catalyzes the processing of primary RNA transcripts. Might have appeared before DNA during evolution.,2.
4、 DNA and RNA syntheses are similar in some aspects but different in others,Similar in fundamental chemical mechanism: both are guided by a template; both have the same polarity in strand extension (5 to 3); both use triphosphate nucleotides (dNTP or NTP). Different aspects: No primers are needed; on
5、ly involves a short segment of a large DNA molecule; uses only one of the two complementary DNA strands as the template strand; no proofreading; subject to great variation (when, where and how efficient to start).,3. The multimeric RNA polymerase in E.coli has multiple functions,The holoenzyme consi
6、sts of five types of subunits (a2bb s)and its is used to synthesize all the RNA molecules in E. coli. The multiple functions include: searches for initiation sites on the DNA molecule and unwinds a short stretch of DNA (initiation); selects the correct NTP and catalyzes the formation of phosphodiest
7、er bonds (elongation); detects termination signals for RNA synthesis (termination).,The E. coli RNA polymerase holoenzyme consists of six subunits: a2bb s.,Possible catalytic subunits,Promoter specificity,Enzyme assembly, promoter recognition, activator binding,Role unknown (not needed in vitro),36.
8、5 kDa,151,155,11 kDa,(32-90 kDa),4. RNA synthesis occurs in a “moving” transcription bubble on the DNA template,Only a short RNA-DNA hybrid (8 bp in bacteria) is present through the transcription process. At each moment, a region of about 17 bp on the E. coli DNA is unwound in the transcription bubb
9、le. The RNA chain is extended at a rate of 50-90 nucleotides/second by the E. coli RNA polymerase. Unwinding ahead of and rewinding behind of the transcription bubble produces positive and negative supercoils respectively on the DNA (relieved by the action of topoisomerases).,5. RNA polymerase recog
10、nizes specific promoter sequences on DNA to initiate transcription,Promoter sequences are located adjacent to genes. Promoters can be identified using “protection assays” (e.g., footprinting techniques). Promoters, although all bind to the same polymerase, have quite variable DNA sequences (surprisi
11、ngly), but with two consensus sequences centered at 10 and 35 positions (the first residue of the RNA is given +1). Promoters having sequences more similar to the consensus are more efficient, and vice versa (from studies of mutations and activity comparison).,The footprinting technique,The footprin
12、t,- protein,+ protein,randomly,Footprinting: Purified RNA polymerase (or other DNA binding protein) is first mixed with isolated and labeled DNA fragment that is believed to bind to the added protein Before that DNA is cut with nonspecific DNase.,In the absence of DNA-binding protein,In the presence
13、 of DNA-binding protein,An actual footprinting result (RNA polymerase binding to a lac promoter),The footprints,Alignment of different promoter sequences from E.coli genes: the 10 (the Pribnow box) and 35 consensus region were revealed.,Sequences of the coding DNA strand is conventionally shown,Add
14、TTTACC N12 TATAAT N7 A,Present only in certain highly expressed genes,Promoter of E.coli Add gene,6. The s subunits enable the E.coli RNA polymerase to recognize specific promoter sites,The RNA polymerase without the s subunit (i.e., the a2bb) is unable to start transcription at a promoter. The s su
15、bunit decreases the affinity of RNA polymerase for general (non-promoter) regions of DNA by a factor of 104. E.coli contains multiple s factors for recognizing different promoters, e.g., s70 for standard promoters; s32 for heat-shock promoters; s54 for nitrogen-starvation promoters. Each type of s f
16、actor allows the cell to coordinately express a set of genes.,Standard Heat-shock Nitrogen starvation,s70 for standard promoters; s32 for heat-shock promoters; s54 for nitrogen-starvation promoters,E.coli contains multiple s factors for recognizing different types of promoters:,7. RNA polymerase unw
17、inds the template DNA then initiate RNA synthesis,The enzyme slides to a promoter region and forms a more tightly bound “closed complex”. Then the polymerase-promoter complex has to be converted to an “open complex”, in which a 12-15 bp covering the region from the AT-rich 10 site to +3 site is unwo
18、und. The essential transition from a “closed” to an “open” complex sets the stage for RNA synthesis, after which the core polymerase moves away from the promoter.,random,8. E.coli RNA polymerase stops synthesizing RNA at specific terminator DNA sequences,Two classes of transcription terminators have
19、 been identified in bacteria: one depends on r protein, the other is r-independent. At the r independent terminator, the transcribed RNA is able to form a stem-loop (palindromic in DNA sequence) structure followed by a stretch of Us (oligoA in DNA). The r-dependent terminator needs the r protein, wh
20、ich has an ATP-dependent RNA-DNA helicase activity, for stopping RNA synthesis.,The r-dependent terminator DNA exhibit no obvious sequence similarities (probably the RNA polymerase detects noncontiguous structural features?). The r-dependent terminator is more often found in phages (where it was ori
21、ginally discovered), but rarely in E.coli. In contrast to what was originally expected, the active signals for stopping RNA synthesis in both r-independent and r-dependent transcription terminators lie in the newly synthesized RNA rather than in the DNA template.,r-independent terminator: a model,Pa
22、lindrome DNA sequences,Oligo Us,Stem-loop (hairpin) structure,Transcription terminator of E.coli Add gene,Model for an r-dependent terminator,9. Transcription is a highly regulated process,Transcription is the first step in the complicated and energy-expensive pathway leading to protein synthesis, a
23、n ideal target for regulating gene expression. The RNA polymerase binds to each promoter in very different efficiency. Protein factors binding to DNA sequences close or distant to the promoters can promote (activator) or repress (repressor) the synthesis of certain RNA molecules.,10. Three kinds of
24、RNA polymerases (I, II, and III) have been revealed for making RNAs in the nuclears of eukaryotic cells,Each is responsible for the transcription of a certain groups of genes: rRNA, mRNA or tRNA genes. The enzymes are often identified by examining their sensitivity towards a-amanitin (from a toxic m
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