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1、Chapter 26 RNA Metabolism1. 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 imp
2、lication of the structural and functional complexity of RNA molecules?1. RNA molecules have great structural and functional diversitylWith structures comparable to proteins in complexity and uniqueness.lFunction as messengers between DNA and polypeptides (mRNA), adapters (tRNA) to match a specific a
3、mino acid with its specific genetic code carried on mRNA, and the structural and catalytic components of the protein-synthesizing ribosomes (rRNA).l Stores genetic information in RNA viruses.lCatalyzes the processing of primary RNA transcripts.lMight have appeared before DNA during evolution. 2. DNA
4、 and RNA syntheses are similar in some aspects but different in otherslSimilar in fundamental chemical mechanism: both are guided by a template; both have the same polarity in strand polarity in strand extension (5 to 3); bextension (5 to 3); both use triphosphate triphosphate nucleotidesnucleotides
5、 (dNTP or NTP).lDifferent aspects: No primersNo primers are needed; only involves a short segment of a large DNA molecule; uses only one of the two complementary DNA strands as the template strand; no proofreadingno proofreading; subject to great variation (when, where and how efficient to start).3.
6、 The multimeric RNA polymerase in E.coli has multiple functionslThe holoenzyme consists of five types of subunits (a2bb s)and its is used to synthesize all the RNA molecules in E. coli.lThe multiple functions include:searches for initiation sites on the DNA molecule and unwinds a short stretch of DN
7、A (initiation);selects the correct NTP and catalyzes the formation of phosphodiester bonds (elongation);detects termination signals for RNA synthesis (termination).The E. coli RNA polymerase holoenzyme consists ofsix subunits: a2bb s.Possible catalytic subunitsPromoterspecificityEnzyme assembly,Enzy
8、me assembly,Enzyme assembly,Enzyme assembly,promoter recognition,promoter recognition,promoter recognition,promoter recognition,activator bindingactivator bindingactivator bindingactivator bindingRole unknownRole unknownRole unknownRole unknown(not needed (not needed (not needed (not needed in vitro
9、in vitroin vitroin vitro) ) ) )36.5 36.5 kDakDa151151 15515511 11 kDakDa( (32-90 32-90 kDakDa) )4. RNA synthesis occurs in a “moving” transcription bubble on the DNA templatelOnly a short RNA-DNA hybrid (8 bp in bacteria) is present through the transcription process.lAt each moment, a region of abou
10、t 17 bp on the E. coli DNA is unwound in the transcription bubble.lThe RNA chain is extended at a rate of 50-90 nucleotides/second by the E. coli RNA polymerase.lUnwinding ahead of and rewinding behind of the transcription bubble produces positive and negative supercoils respectively on the DNA (rel
11、ieved by the action of topoisomerases).5. RNA polymerase recognizes specific promoter sequences on DNA to initiate transcriptionlPromoter sequences are located adjacent to genes.lPromoters can be identified using “protection assays” (e.g., footprinting techniques). lPromoters, although all bind to t
12、he same polymerase, have quite variable DNA sequences (surprisingly), but with two consensus sequences centered at 10 and 35 positions (the first residue of the RNA is given +1).lPromoters having sequences more similar to the consensus are more efficient, and vice versa (from studies of mutations an
13、d activity comparison).The The footprintingfootprintingtechniquetechniqueThe footprint- protein+ proteinrandomlyFootprinting: Purified RNA polymerase (or other DNA binding protein) is firstmixed with isolated and labeled DNA fragment that is believed to bind to the added proteinBefore that DNA is cu
14、t with nonspecific DNase.In the absence of DNA-binding proteinIn the presence of DNA-binding proteinAn actual footprinting result (RNA polymerasebinding to a lac promoter)The footprintsAlignment of different promoter sequences fromE.coli genes: the 10 (the Pribnow box) and 35 consensus region were r
15、evealed.Sequences of the coding DNA strandis conventionallyshownAdd TTTACC N12 TATAAT N7 A Present only in certain highly expressed genesPromoter of E.coli Add gene6. The s s subunits enable the E.coli RNA polymerase to recognize specific promoter siteslThe RNA polymerase without the s subunit (i.e.
16、, the a2bb) is unable to start transcription at a promoter.lThe s subunit decreases the affinity of RNA polymerase for general (non-promoter) regions of DNA by a factor of 104.lE.coli contains multiple s factors for recognizing different promoters, e.g., s s70 for standard promoters; s s32 for heat-
17、shock promoters; s s54 for nitrogen-starvation promoters.lEach type of s factor allows the cell to coordinately express a set of genes.StandardHeat-shockNitrogenstarvations s70 for standard promoters; s s32 for heat-shock promoters; s s54 for nitrogen-starvation promotersE.coli contains multiple s s
18、 factors for recognizing different types of promoters:7. RNA polymerase unwinds the template DNA then initiate RNA synthesislThe enzyme slides to a promoter region and forms a more tightly bound “closed complex”.lThen the polymerase-promoter complex has to be converted to an “open complex”, in which
19、 a 12-15 bp covering the region from the AT-rich 10 site to +3 site is unwound.lThe 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.random8. E.coli RNA polymerase stops synthesizing RNA at specif
20、ic terminator DNA sequenceslTwo classes of transcription terminators have been identified in bacteria: one depends on r protein, the other is r-independent.lAt the r r independent terminator, the transcribed RNA is able to form a stem-loop (palindromic in DNA sequence) structure followed by a stretc
21、h of Us (oligoA in DNA).lThe r r-dependent terminator needs the r r protein, which has an ATP-dependent RNA-DNA helicase activity, for stopping RNA synthesis.lThe r-dependent terminator DNA exhibit no obvious sequence similarities (probably the RNA polymerase detects noncontiguous structural feature
22、s?).lThe r-dependent terminator is more often found in phages (where it was originally discovered), but rarely in E.coli.lIn 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 synthe
23、sized RNA rather than in the DNA template.r r-independentterminator: a modelPalindrome DNA sequences Oligo UsStem-loop (hairpin) structureTranscription terminator of E.coli Add geneModel for an r r-dependent terminator9. Transcription is a highly regulated processlTranscription is the first step in
24、the complicated and energy-expensive pathway leading to protein synthesis, an ideal target for regulating gene expression.lThe RNA polymerase binds to each promoter in very different efficiency.lProtein factors binding to DNA sequences close or distant to the promoters can promote (activator) or rep
25、ress (repressor) the synthesis of certain RNA molecules.10. Three kinds of RNA polymerases (I, II, and III) have been revealed for making RNAs in the nuclears of eukaryotic cellslEach is responsible for the transcription of a certain groups of genes: rRNA, mRNA or tRNA genes.lThe enzymes are often i
26、dentified by examining their sensitivity towards a-amanitin (from a toxic mushroom). Eukaryotic RNA polymerases11. RNA polymerase II (Pol II) binds to promoters of thousands of protein-coding geneslMany Pol II promoters contain a TATAAA sequence (called a TATA box) at -30 position and an initiator s
27、equence (Inr) at +1 position.lThe preinitiation complex (including Pol II) is believed to assemble at the TATA box, with DNA unwound at thelInr sequence.lHowever, many Pol II promoters lack a TATA or Inr or both sequences!General features of promoters for protein-codinggenes in higher eukaryotesGCbo
28、xCAATbox12. Pol II is helped by an arrays of protein factors (called transcription factors) to form an active transcription complex at a promoter lFirst the TATA-binding protein (TBP) binds to the TATA box, then TFIIB, TFIIF-Pol II, TFIIE, and TFIIH will be added in order forming the closed complex
29、at the promoter.lTFIIH then acts as a helicase to unwind the DNA duplex at the Inr site, forming the open complex.lA kinase activity of TFIIH will phosphoryate the C-terminal domain (CTD) of Pol II, which will initiate RNA synthesis and release the elongation complex.lTFIIE and TFIIH will be release
30、d after the elongation complex moves forward for a short distance.lElongation factors will then join the elongation complex and will suppress the pausing or arrest of the Pol II-TFIIF complex, greatly enhancing the efficiency of RNA synthesis.lThe termination of transcription of Pol II happens by an
31、 unknown mechanism.lThis basal process of initiating RNA synthesis by Pol II is elaborately regulated by many cell or tissue specific protein factors that will binds to the transcription factors, mostly act in a positive way.lWhen Pol II transcription stalls at a site of DNA lesion, TFIIH will binds
32、 at the lesion site and appears to recruit the entire nucleotide-excision repair complex. TBPDNAA proposed modelfor Pol II- catalyzedmRNA synthesis13.The action of RNA polymerases can be specifically inhibitedlThree-ring-containing, planar antibiotic molecules like actinomycin D intercalates between
33、 two successive GC base pairs in duplex DNA, preventing RNA polymerases (all types) to move along the template (thus the elongation of RNA synthesis).lRifampicin (an antibiotic) binds to the b subunit of bacterial RNA polymerases, preventing the initiation of RNA synthesis.la a-amanitin blocks eukar
34、yotic mRNA synthesis by binding to RNA polymerase II.14. RNA molecules are often further processed after being synthesized on the DNA templatelThe primary transcripts of eukaryotic mRNAs are often capped at the 5 end, spliced in the middle (introns removed and exons linked), polyadenylated at the 3
35、end.lThe primary transcripts of both prokaryotic and eukaryotic tRNAs are cleaved from both ends, spliced in some cases, and modified for many of the bases and sugars.15. An eukaryotic mRNA precursor acquire a 5 cap shortly after transcription initiateslA GMPGMP component (from a GTP) is joined to t
36、he 5 end of the mRNA in a novel 5,5-5,5-triphosphate triphosphate linkage.linkage.lThe guanine base is then methylatedmethylated at the N-7.lThe 2-OH groups of the 1st and 2nd nucleotides adjacent to the 7-7-methylguanine methylguanine capcap may also be methylated in certain organisms.lThe methyl g
37、roups are transferred from S-S-adenosylhomocysteineadenosylhomocysteine.A 5 cap is addedA 5 cap is addedto to eukaryotic eukaryotic mRNAs mRNAs Before Before transcriptiontranscriptionendsendsThe 5 cap foundThe 5 cap foundon the on the eukaryoticeukaryoticmRNAsmRNAs16. Most eukaryotic mRNAs have a p
38、oly(A) tail at the 3endlThe tail consists of 80 to 250 adenylate residues.lThe mRNA precursors are extended beyond the site where poly(A) tail is to be added.lAn AAUAAA sequence was found to be present in all mRNAs and marks (together with other signals at the 3end) the site for cleavage and poly(A)
39、 tail addition (11 to 30 nucleotides on the 3end of the AAUAAA sequence).lThe specific endonuclease and polyadenylate polymerase, and other proteins probably exist as a multiprotein complex to catalyze this event.A poly(A) tailis usually addedat the 3 end of an mRNA moleculevia a processing step.17.
40、 EM studies of mRNA-DNA hybrids revealed the discontinuity of eukaryotic geneslEach gene was found to be a continuous fragment of DNA in the bacterial genome.lBut Berget and Sharp (1977) observed single-stranded DNA loops when examining adenovirus mRNA-DNA hybrids by electron microscopy.lSuch single
41、-stranded DNA loops was widely observed when examining such RNA-DNA hybrids.lIntron sequences were proposed to be present on the template DNA sequences, which are removed during RNA processing, with exons linked together precisely.lAlmost all genes in vertebrates contain introns (but histone genes d
42、oes not).lMany genes in certain yeasts do not contain introns.lIntrons are also found in a few bacterial and archaebacterial genes (but far less common than in eukaryotic cells).EM studies of mRNA-DNA hybrids for thechicken ovalbumin gene (the R-looping technique)18. Four classes of introns have bee
43、n revealed having different splicing mechanismslGroup I introns are found in some nuclear, mitochondrial and chloroplast genes encoding rRNAs, mRNAs, and tRNAs.lGroup II introns are often found in genes encoding mRNAs in mitochondrial and chloroplast DNA of fungi, algae, and plants.lGroup III intron
44、s (the largest group) are found in genes encoding eukaryotic nuclear mRNAs.lGroup IV introns are found in genes encoding the tRNAs in the nuclear genomic DNA of eukaryotes19. Group I introns are self-splicing and use a guanine nucleoside or nucleotide as the cofactorlThe intron present in the rRNA p
45、recursor of Tetrahymena was found to be removed by itself without using any proteins (Thomas Cech, 1982).lThe intron is removed and the two exons precisely linked via two nucleophilic transesterification reactions (with two 3-OH group act as the nucleophiles).Group I Group I intronsintronsare remove
46、d byare removed byself-splicing viaself-splicing viatwo two nucleophilicnucleophilictransesterificationtransesterificationreactions.reactions.The predicted secondarystructure of the self-splicingrRNA intron of TetrahymenaThe internal guide sequence5 splice site3 splice site20. Group II introns also
47、undergo self-splicing using forming a lariat-like intermediate lBut the 2-OH group of an adenylate residue within in removing intron played the role of the 3-OH group of the guanine nucleoside or nucleotide in group I intron self-splicing.Group II introns areremoved via self-splicing with an adenyla
48、te residueof the removing intron acts as thenucleophile,formingan lariate-like Intermediate.21. Type III introns are found in the nuclear mRNA primary transcripts and have the largest numberslThe splicing exon-intron junctions, determined by comparing the sequences of the genomic DNA with that of th
49、e cDNA prepared from the corresponding mRNA, in mRNA precursors are specified by sequences at the two ends of the introns: begin with GU and end with AG.lType III introns are removed via a very similar way as that of type II introns except being helped by several highly conserved small nuclear ribon
50、ucleoproteins (snRNPs), each containing a class of U-rich small nuclear RNAs (snRNAs).Type III Type III intronsintrons, found on nuclear , found on nuclear mRNA mRNA primaryprimarytranscripts, are removed via the transcripts, are removed via the spliceosomesspliceosomes22. group IV introns are found
51、 in tRNA precursors and are removed by endonuclease and RNA ligaselThe splicing endonuclease first cleaves the phosphodiester bonds at both ends of the intron.lATP is needed for the RNA ligase activity to join the two exons.lThe joining reaction is similar to the DNA ligase-catalyzed reaction.lThe m
52、echanism of cleaving group IV introns is different from that of group I, II, and III introns, all including two transesterification reactions.Group IV Group IV introns introns arearespliced via the spliced via the action of specificaction of specificendonucleaseendonuclease and andRNA RNA ligaseliga
53、se. .RNARNA ligase ligase23. Alternative proteins may be produced from one single gene via differential RNA processinglThe multiple transcripts produced from such a gene may have more than one site for cleavage and polyadenylation (as for immunoglobulin heavy chains), alternative splicing (as for th
54、e myosin heavy chains in fruit flies), or both (as for the calcitonin gene in rats).lIn different cells or at different stages of development, the transcript may be processed differently to produce different gene products (proteins).Multiple Multiple mRNAs mRNAs (thus polypeptide (thus polypeptide c
55、hains) can be produced via chains) can be produced via differential RNA processing.differential RNA processing.24. The different rRNA molecules of both prokaryotes and eukaryotes are generated from single pre-rRNAslThe 16S, 23S and 5S rRNAs (together with certain tRNAs) in bacteria are all generated
56、 from a single 30S pre-rRNA (about 6.5 kb, transcribed by RNA polymerase I).lThere are seven pre-rRNA genes in the E.coli genome (each encoding a different tRNA).lThe 18S, 28S and 5.8S rRNAs in eukaryotes are generated from a single 40S pre-rRNA (14 kb).lThe 5S rRNA in eukaryotic cells is generated
57、separately (transcribed by RNA polymerase III).All the All the rRNAs rRNAs are derivedare derivedfrom a single precursor infrom a single precursor inprokaryotic cells.prokaryotic cells.The 18S, 5.8S, and 28S The 18S, 5.8S, and 28S rRNAs rRNAs in in eukaryotic eukaryotic cells are cells are derived f
58、rom one pre-derived from one pre-rRNA rRNA molecule (the processingmolecule (the processingneeds small needs small nucleolar nucleolar RNA-containing proteins).RNA-containing proteins).25. Primary tRNA transcripts undergo a series of posttranscriptional processinglThe extra sequences at the 5 and 3
59、ends are removed by RNase P and RNase D respectively. lThe RNA in RNaseP is catalytic (Altman, 1983)lType IV introns are occasionally present in pre-tRNAs in eukaryotic cells.lThe CCA sequence is generated at the 3 end by the action of tRNA nucleotidyltransferase (having three active sites for the t
60、hree ribonucleotides added).lSome of the bases in tRNA molecules are modified by methylation, deamination, reduction and others.The processing of the primary tRNA transcripts include removal of the 5and 3 ends, addition of the CCA sequenceat the 3 end, modification of many bases,and splicing of intr
61、ons (in eukaryotic cells).Some typical modificied bases foundIn mature tRNA molecules.26. More RNA molecules (ribozymes) were found to be catalyticlCatalytic RNA molecules were also found in the virusoid RNA (called hammerhead ribozymes).lRNAs in the spliceosomes (the U-rich RNAs) and ribosomes are
62、also believed to be catalytic.lA specific 3-D structure is required for ribozymes to be catalytic.lRibozymes often orient their substrates via base pairing.lThe excised intron (414 nucleotides) of the pre-rRNA of Tetrahymena is further processed to a RNA fragment of 395 nucleotides named as L-19 IVS
63、;(intervening sequence lacking 19 nucleotides)lA portion of the internal guide sequence remains at the 5 end of L-19 IVS and the guanosine binding site is still intact.lDr. Cech reasoned that L-19 IVS might act on external substrates.lL-19 IVS is able to catalyze the lengthening of some oligonucleot
64、ides, like a (C)5 oligomer, at the expense of others (being both a nuclease and polymerase).L-19 IVS functions as a real catalyst in the test tubeIncubation time (minutes)Labeled substrateRNANucleaseactivityRNApolymeraseactivityThe M1 RNAin ribonucleaseP is catalyticThe intron inthe pre-rRNA ofTetra
65、hemena is self-spliced27. The cellular mRNAs are degraded at different rateslThe level of a protein in a cell is determined to some extent by the level of its mRNA, which depends on a balance of the rates on its synthesis and degradation.lThe half of lives of different mRNA molecules vary greatly, f
66、rom seconds to many cell generations.l l3 hairpin and poly(A) tails have been shown to increase halve lives of mRNAs, but multiple, sometimes overlapping AUUUA sequences have been shown to decrease halve lives. lThe 5 3 exoribonuclease is probably the major degrading enzyme for mRNAs.lThe polynucleo
67、tide phosphorylase may be another enzyme degrading mRNAs.lPolynucleotide phosphorylase was used to synthesize RNA for the first time in the test tube (Severo Ochoa shared the Nobel Prize with Arthur Kornberg in 1959 for this discovery).l(NMP)n+1 + Pi(NMP)n + NDPlThis enzyme was used to synthesize RN
68、A polymers of different sequences and frequencies of bases for the elucidation of the genetic codes.Average half lives of mRNA moleculesBacteria1.5 minutesVertebrates 3 hours 28. Reverse transcriptases catalyze the production of DNA from RNAlThe existence of this enzyme in retroviruses ( RNA viruses
69、) was predicted by Howard Temin in 1962, and proved by Temin and David Baltimore in 1970.lThis enzyme catalyzes three reactions: RNA-directed DNA synthesisRNA-directed DNA synthesis using tRNAs as primers; Degradation of the RNA templateDegradation of the RNA template; DNA-directed DNA synthesisDNA-
70、directed DNA synthesis;lThe enzyme has no 3 5 proofreading exonuclease activity, thus generating high rate of mutations.lThis enzyme is widely used to synthesize complementary DNAs (cDNAs) from mRNAs.Reverse transcriptases catalyzes thesythesis of DNA from RNA template.29. Telomerase catalyzes the s
71、ynthesis of the repeating telomere sequences (TxGy) using an internal RNA templatelTelomeres consist of a few to a large number of tandem copies of a short oligonucleotide sequence that are located at the two ends of the linear chromosomal DNAs, having a 3 single strand extension (on the TG strand).
72、l Telomerase acts to prevent the chromosomal ends from becoming shortened after each replication (the end part of the lagging strand can not be duplicated).lTelomerase is actually a reverse transcriptase, but uses a short segment of an internal RNA molecule (150 nucleotides) as the template to exten
73、d the end.lThe CyAx strand (the lagging strand) is believed to be synthesized by a DNA polymerase using a RNA primer.lThe ends of a linear chromosome is often protected by binding to specific proteins, forming a T loop structure in higher eukaryotes, where the single-stranded DNA is sequestered.lThe
74、 length of the telomere seems to be inversely related to the life span of cells and individuals (shortens as one ages).Problem posed in the replicationof linear DNA:the end of onedaughter strandwill be shortenedafter each round of replication.The “inchworm”(尺蠖）尺蠖）model fortelomerase actionThe T loop
75、 observed atone end of a mammalianchromosome.30. Some viral RNAs are replicated by RNA-directed RNA polymeraselThe RNA genomes of some viruses (having bacteria, animals or plants as their hosts) are replicated using RNA-directed RNA polymerases (or RNA replicases).lThe RNA replicase from bacteriopha
76、ge-infected E.coli cells consists of subunits encoded both by the viruses and the host genome. lThey have features similar to that of DNA-directed RNA polymerases, but are usually specific for the RNA of the specific viruses.lRNA replicases do not have proofreading activities.31. Self-replicating RN
77、A molecules might be important for life to be produced at the very beginninglThe realization of the structural and functional complexity of RNA led a few scientists to propose in 1960s that RNA might have serves as both information carrier and catalyst at the early stage of evolution.lSynthesis of t
78、he peptide bonds of proteins seems to be catalyzed by the rRNA component of ribosomes.lA self-replicating mechanism for a RNA molecule can be proposed based on the studies of the self-splicing process of group I introns.lSELEX (systematic evolution of ligands by exponential enrichment) techniques ha
79、ve been used to select RNA molecules (from a random RNA library) that bind to various biomolecules (including amino acids, organic dys, nucleotides, cyanocobalamin and others).A model to explain the RNA-dependent synthesisof an RNA polymer from oligonucleotide precursors.An ATP-bindingRNA was genera
80、tedand isolated using SELEX.SummarylTranscription shares the basic chemical mechanisms with replication except: no primers required, no proofreading activity exist.lTranscription begins at specific promoter sequences (which can be identified using footprinting technique) and ends at specific termina
81、tor sequences (being r-independent or dependent in bacteria, and not well understood in eukaryotes).lOne multimeric RNA polymerase catalyzes the synthesis of all the RNA molecules in bacteria and different RNA polymerases are used to synthesize the different types of RNAs in eukaryotes. lPrimary RNA
82、 transcripts are further processed: capped, tailed, spliced and sometimes edited for the mRNAs; ends removed and modified, bases modified for the tRNAs; cleaved and sometimes spliced for the rRNAs.lCatalytic RNAs were discovered when studying RNA processing (splicing of group I and II introns, remov
83、al of the 5end of pre-tRNAs).lRNAs can act as real enzymes (L-19 IVS, hammerhead ribozymes).lDNA can be synthesized by using RNA as templates in a reaction catalyzed by reverse transcriptase.lThe telomeres of eukaryotic chromosomes are synthesized by the action of telomerase, using an RNA as template.lRNA replicase catalyzes the synthesis of RNA from RNA templates.lRNA is very likely the first type of biomacromolecules produced during biochemical evolution.
