Substrate binding and catalysis by ribonuclease P from cyanobacteria and Escherichia coli are affected differently by the 3′ terminal CCA in tRNA precursors

P oc. Na l. Acad. Sci. USA Vol. 96, pp. 6672–6677, June 1999 Biochemis y Subs a e binding and ca alysis by ibonuclease P om cyanobac e ia and Esche ichia coli a e a ec ed di e en ly by he 3* e minal CCA in RNA p ecu so s ALBERTO PASCUAL AND AGUSTI ´NVIOQUE* Ins i u o de Bioquı´mica Vege al y Fo osı´n esis, Cen o de In es igaciones Cien ı´ icas Isla de la Ca uja, Uni e sidad de Se illa-Consejo Supe io de In es igaciones Cien ı´ icas, A enida Ame ico Vespucio syn, 41092 Se ille, Spain Communica ed by Sidney Al man, Yale Uni e si y, New Ha en, CT, Ap il 7, 1999 ( ecei ed o e iew Janua y 15, 1999) ABSTRACT We ha e s udied he e ec o he 3* e minal CCA sequence in p ecu so s o RNAs on ca alysis by he RNase P RNA o he holoenzyme om he cyanobac e ium Synechocys is sp. PCC 6803 in a comple ely homologous sys- em. We ha e ound ha he absence o he 3* e minal CCA is no de imen al o ac i i y, which is in sha p con as o wha is known in o he bac e ial sys ems. We ha e ound ha his is also ue in o he cyanobac e ia. This si ua ion co e- la es wi h he anomalous s uc u e o he J15y16 loop in cyanobac e ia, which is an impo an loop in he CCA in e - ac ion in Esche ichia coli RNase P, and wi h he ac ha cyanobac e ia do no code he CCA sequence in he genome bu add i pos ansc ip ionally. Modi ica ion o nucleo ides 330–332 in he J15y16 loop o Synechocys is RNase P RNA om GGU o CCA has a modes e ec on k ca o CCA- con aining subs a es and has no e ec on clea age-si e selec ion. We ha e de eloped a di ec physical assay o he in e ac ion be ween RNase P RNA and i s subs a e, which was immobilized on a il e , and we ha e de e mined ha Synechocys is RNase P RNA binds wi h be e a ini y he subs a e lacking CCA han he subs a e con aining i . Ou esul s indica e a mode o subs a e binding in RNase P om cyanobac e ia ha is di e en om binding in o he eubac- e ia and in which he 3* e minal CCA is no in ol ed. RNase P is a ubiqui ous enzyme esponsible o gene a ing he 59end o p e- RNAs by a single endonucleoly ic clea age (1, 2).Inbac e ia, he enzyme iscomposed o an RNAsubuni and a p o ein subuni . The RNA subuni is he ca aly ic componen and, unde app op ia e condi ions in i o, i can clea e subs a es in he absence o he p o ein (3). In addi ion o all he p e- RNAs, se e al o he na u al and a i icial subs a es a e ecognized by RNase P (4–7). Se e al s udies ha e exam- ined how a single enzyme, gene ally om Esche ichia coli and Bacillus sub ilis, can ecognize so many di e en subs a es and clea e all o hem a he co ec posi ion ( e iewed in e s. 2, 8, and 9). One o he main conclusions om hese s udies is he c ucial ole played by he 39 e minal RCCA sequence o p e- RNAs, whe e he unde lined nucleo ides in e ac wi h a GGU sequence in he loop connec ing helices P15 and P16 (loop J15y16) (10–12). In addi ion, he 39 e minal CCA pa icipa es in binding o Mg 21 ions used in ca alysis (13). This in e ac ion is impo an in de ining he clea age si e and eac ion a e. In cyanobac e ia, he J15y16 loop has a s uc u e ha de ia es om he consensus, and i gene ally lacks a GGU sequence (Fig. 1) (14, 15). In some ins ances, he e is an ex a helix inse ed in his loop. We ha e in es iga ed he ole o he 39 e minal CCA in cyanobac e ial p e- RNAs on clea age by cyanobac e ial RNase P RNA and he holoenzyme. Kine ic analyses indica e ha cyanobac e ia RNase P has no p e e - ence o CCA-con aining subs a es, as does he E. coli en- zyme, unde single- o mul iple- u no e condi ions. The Synechocys is RNase P RNA con ains a GGU sequence in he J15y16 loop. We ha e analyzed how mu agenesis o his sequence a ec s ac i i y. The ela i e a ini ies o Synechocys- is RNase P RNA o p e- RNAs con aining o lacking he 39 e minal CCA ha e been analyzed by a me hod based on he immobiliza ion o he p e- RNAs on a memb ane. This p o- cedu e acili a es he de ec ion o he RNA–RNA in e ac ions when he binding a ini y is oo low o be de ec ed by gel e a da ion. Ou esul s indica e ha cyanobac e ial RNase P binds and eac s on subs a es lacking he 39 e minal CCA. These esul s migh e lec he ac ha in cyanobac e ia he 39 e minal CCA gene ally is no encoded in he genome bu is added pos ansc ip ionally, and hey suppo he ecen sugges ion ha he e is a co ela ion o he p ima y s uc u e o RNA genes wi h RNase P RNA s uc u e and subs a e p e e ence (16). MATERIALS AND METHODS P epa a ion o RNAs. RNAs we e p epa ed by in i o un-o ansc ip ion wi h T7 RNA polyme ase om empla e plasmids as desc ibed (17). A e ansc ip ion, he RNAs we e dephospho yla ed wi h cal in es inal phospha ase and labeled a he 59end wi h polynucleo ide kinase and [ g - 32 P]ATP. Al e na i ely, when uni o mly labeled RNA was equi ed, [ a - 32 P]CTP was included in he eac ion mix. Templa e plasmids o in i o ansc ip ion o E. coli M1 RNA (17), Synechocys is sp. PCC 6803 and Anabaena sp. PCC 7120 RNase P RNAs (14), and pGln (p ecu so o Synecho- cys is RNA Gln ) (18) al eady ha e been desc ibed. Pseudan- abaena sp. PCC 6903 RNase P was ob ained om a empla e p epa ed by C. Tous (Se illa Uni e si y) on he basis o he published sequence (19). Plasmid pT7Glu was p epa ed o he syn hesis o Synecho- cys is 6803 pGlu (p ecu so o Synechocys is RNA Glu ). The Synechocys is 6803 nE gene was ampli ied by PCR om genomic DNA wi h p ime s based on he published sequence (20). The o wa d p ime (59-CGACGGGATCCTAAATAC- GACTCACTATAGTCTGAAATAACGAACTG-39) con- ains a BamHI si e and he T7 p omo e sequence and o e laps he 59end o he p e- RNA sequence. The e e se p ime (59-GTCCCAAGCTTGGATGGACGCCTGGTACCCCCA- AGGGAA-39) o e laps he 39end o p e- RNA sequence and The publica ion cos s o his a icle we e de ayed in pa by page cha ge paymen . This a icle mus he e o e be he eby ma ked ‘‘ad e isemen ’’ in acco dance wi h 18 U.S.C. §1734 solely o indica e his ac . PNAS is a ailable online a www.pnas.o g. Abb e ia ions: p e- RNA, p ecu so o RNA; pGlu, p ecu so o Synechocys is RNA Glu ; pGln, p ecu so o Synechocys is RNA Gln ; pGlnCCA, p ecu so o Synechocys is RNA Gln con aining he 39 e - minal CCA. *To whom ep in eques s should be add essed. e-mail: ioque@ cica.es. 6672 con ains a FokI,aBs NI, and a HindIII si e. A PCR p oduc o he expec ed size was pu i ied, diges ed wi h HindIII and BamHI, and liga ed in o pUC19 ea ed wi h he same en- zymes. The clone was con i med by sequencing. P e- RNAs we e p epa ed by T7 RNA polyme ase-based un-o ansc ip ion by using as empla es pT7Gln o pT7Glu diges ed wi h ei he FokI ( o gene a e subs a es lacking he 39 e minal CCA) o Bs NI ( o gene a e subs a es con aining he 39 e minal CCA). Fig. 2 shows he p edic ed seconda y s uc u es o Synechocys is pGln and pGlu. Pu i ica ion o P o eins. RNase P p o ein om E. coli (C5 p o ein) was pu i ied om cul u es o BL21(DE3) ca ying plasmid pARE7 as desc ibed (17). RNase P p o ein om Synechocys is was pu i ied om cul u es o BL21(DE3) ca y- ing plasmid pARA2 as desc ibed (18). RNase P Ac i i y Assays. RNase P RNA assays we e done in 50 mM T iszHCl, pH 7.5y100 mM MgCl 2 y1 M KCl. RNase P RNAs we e incuba ed in assay bu e o 5 min a 37°C be o e addi ion o subs a e. Holoenzyme was econs i u ed by di ec mixing o RNase P RNA wi h a 10- old mola excess o pu i ied RNase P p o ein in assay bu e and incuba ed o 5 min a 37°C be o e addi ion o subs a e. E. coli holoenzyme assays we e done in 50 mM T iszHCl, pH 7.5y10 mM MgCl 2 y400 mM NH 4 Cly0.1% T i on X-100 (21). Synechocys is 6803 holoen- zyme assays we e done in 50 mM T iszHCl, pH 7.5y50 mM MgCl 2 . These condi ions ha e been ound o be op imal o he Synechocys is holoenzyme (unpublished da a). Aliquo s o he eac ions we e mixed a di e en imes wi h loading dye con aining u ea and EDTA. The amoun o p ocessing was es ima ed by sepa a ing eac ion p oduc s on ac ylamideyu ea gels and quan i ica ion wi h a Cyclone Phospho Sys em (Packa d). Kine ic cons an s we e es ima ed om double- ecip ocal plo s o ini ial eac ion a es a di e en subs a e concen a ions as desc ibed (22). Subs a e concen a ion was a ied in he ange o 0.01–100 m M. Enough da a we e collec ed o each enzyme-subs a e pai o ha e a educed e o in he es ima ion o ca aly ic cons an s shown in Table 1. Si e-Di ec ed Mu agenesis. Mu a ions G330C, G331C, and U332A we e in oduced simul aneously in he Synechocys is npB gene by si e-di ec ed mu agenesis wi h he oligonucleo- ide 59-CCTGCCCCATGATTCCAGGAACCGCTTGAGG- AATTTGG-39. The U.S.E. Mu agenesis Ki om Pha macia was used o his pu pose. Analysis o RNA—RNA A ini y. Gel e a da ion o p e- RNA wi h RNase P RNAs was done as desc ibed (23). Fil e -bound in e ac ion analysis was pe o med by ans- e ing equal amoun s o he p e- RNAs o be analyzed o a memb ane (Hybond N 1 , Ame sham) wi h a slo –blo appa- a us. The RNAs we e ixed o he memb ane by baking o 2 h a 80°C, and hen he il e was blocked by soaking o 2ha oom empe a u e in 20 ml o 20 mM HepeszKOH, pH 8.0y100 MgCl 2 y1 M KCly2 mg/ml yeas RNA (R-6625; Sigma), which was p epa ed as desc ibed (24). A e he blocking s ep, he il e was soaked o 20 min in a minimal olume o he same solu ion, bu wi hou yeas RNA, con aining 10,000 cpmyml o 32 P-labeled RNase P RNA. The il e was washed wice o 20 min wi h he same solu ion wi hou yeas RNA, and he bound adioac i i y was quan i ied wi h a Cyclone Phospho Sys em. The in ensi y o he signal was p opo ional o he amoun o p e- RNA bound o he il e in he ange o 2–200 pmol. We ound ha no binding o p e- RNA is de ec ed when he RNase P RNA is immobilized on he il e ins ead o he p e- RNA. RESULTS E ec o 3*Te minal CCA in P e- RNA on RNase P Clea age Kine ics. The p o ein and RNA subuni s o RNase P, as well as a p e- RNA subs a e, om he cyanobac e ium Synechocys is 6803 ha e been cloned and cha ac e ized (15, 18). The e o e, a comple ely homologous sys em o in es iga e he enzyme—subs a e in e ac ion du ing RNase P ca alysis in cyanobac e ia is a ailable. We ha e looked a he e ec ha he 39 e minal CCA sequence in he subs a e has on clea age a e and subs a e binding. Fi s , we analyzed clea age a e unde single- u no e condi ions (p e-s eady-s a e condi ions) o de e mine he e ec o he CCA sequence on he a e o he chemical clea age eac ion. In his way he eac ion a e was no a ec ed by slow p oduc elease ha has been shown o limi he o e all eac ion a e unde mul iple- u no e condi- ions in E. coli. Fig. 3 shows ha pGln was clea ed sligh ly as e han pGlnCCA (p ecu so o Synechocys is RNA Gln con aining he 39 e minal CCA) by Synechocys is RNase P FIG. 1. S uc u e o he J15y16 loop in he RNase P RNAs used in his wo k. The J15y16 loop, connec ing helices P15 and P16, is shown om le o igh o E. coli,Synechocys is 6803, Anabaena 7120, and Pseudanabaena 6903. The p oposed (11) base pai ing be ween he sequence RCCA-39 in p e- RNAs and a conse ed GGU sequence in J15y16 o E. coli is shown. The GGU sequence mu a ed in Synechocys is o CCA o gene a e mu an CCA332 is boxed. FIG. 2. S uc u e o p e- RNA subs a es used in his wo k. These subs a es could be p epa ed by in i o ansc ip ion ei he con aining o lacking he 39 e minal CCA (boxed), depending on he es ic ion enzyme used o diges he empla e, as desc ibed in he ex . Biochemis y: Pascual and Vioque P oc. Na l. Acad. Sci. USA 96 (1999) 6673 RNA o holoenzyme, whe eas E. coli RNase P RNA o holoenzyme had a clea p e e ence o pGlnCCA. The p e - e ence o he E. coli enzyme o CCA-con aining subs a es is well documen ed (23, 25, 26). Synechocys is RNase P RNA beha ed in an opposi e way. This esul was no a peculia i y o he Synechocys is enzyme o o he pGln subs a e. RNase P RNAs om he cyanobac e ia Anabaena 7120 and Pseudan- abaena 6903 showed a simila beha io (Fig. 4A). Also, when a di e en subs a e was used, Synechocys is pGlu, a simila esul was ob ained (Fig. 4C). E. coli RNase P RNA had a clea p e e ence o he CCA-con aining subs a e, whe eas he Synechocys is RNase P RNA clea ed he CCA-lacking sub- s a e sligh ly be e . Synechocys is is one o he ew cyanobac e ia ha con ain a GGU sequence in J15y16. Bu as has been discussed p e iously (14), J15y16 has a s uc u e in Synechocys is ha di e s om ha in E. coli. To in es iga e he ole o J15y16 o Synechocys is on enzyma ic ac i i y, we ha e changed he GGU sequence o CCA by si e-di ec ed mu agenesis o gene a e mu an CCA332. Unde single- u no e condi ions, RNase P RNA CCA332 had he same subs a e p e e ence han wild ype (Fig. 4A). Holoenzyme econs i u ed wi h RNA CCA332 also beha es simila ly (Fig. 4B). RNA CCA332 has a eac ion a e abou 50% o he wild ype in he RNA-alone eac ion and abou 20% lowe o he wild ype in he holoenzyme eac ion. The holoenzyme ac i i y o RNA CC332 could be inc eased up o 50% o wild ype by inc easing he p o ein concen a ion (no shown). The e o e, i seemed ha RNA CCA332 was pa ially de ec i e in i s in e ac ion wi h he p o ein, and, he eby, i equi ed highe p o ein concen a ions han wild ype o each maximum ac i i y. The p esence o absence o CCA in he subs a e had no e ec on he si e o clea age. The modi ica ion o J15y16 in mu an CCA332 also had no e ec on he si e o clea age. Fig. 4Dshows ha in all cases he 59 agmen had he same size, which co esponded o clea age a he 11 posi ion, as de e mined by p ime ex ension (18) (A.V., unpublished da a). The esul s ob ained unde single- u no e condi ions we e con i med ully unde mul iple- u no e assay condi ions. Kine ic cons an s o Synechocys is wild- ype and mu an CCA332 RNase P RNAs we e de e mined unde mul iple- u no e condi ions (s eady s a e) o subs a e con aining and lacking he 39 e minal CCA (Table 1). Fo compa ison, E. coli M1 RNA also was analyzed. The e ec s o he 39 e minal CCA on K m and k ca o M1 RNA we e simila o wha has been desc ibed p e iously o a numbe o subs a es. K m was 23- old lowe o he CCA-con aining subs a e. k ca also was lowe (13- old) because o he lowe p oduc elease a e o he CCA-con aining subs a e. Synechocys is RNase P RNA had a K m o pGln simila o ha o he E. coli M1 RNA, bu he K m o pGlnCCA was mo e han 100- old highe . The p esence o CCA had a de imen al e ec on K m , which was he opposi e o wha happens in E. coli. Con e sely, k ca was 120- old highe o pGlnCCA han o pGln. The mu a ion in J15y16 in RNase P RNA CCA332 had no signi ican e ec on K m ; i showed jus a 3- old educ ion o he K m o pGlnCCA. The k ca o pGlnCCA was educed 13- old. RNase P RNA—P e- RNA In e ac ion. The esul s de- sc ibed in he p e ious sec ion indica ed ha he 39 e minal CCA in p e- RNA had an e ec on RNase P ac i i y in cyanobac e ia ha was di e en om wha was desc ibed p e iously in o he bac e ial RNase P RNAs. The K m was inc eased d as ically when he 39 e minal CCA was p esen in he subs a e. The e o e, we decided o s udy whe he his e ec was due o a de ec in enzyme–subs a e a ini y. I is well es ablished ha E. coli M1 RNA has highe a ini y o CCA-con aining subs a es. This has been shown mainly by gel- e a da ion assays (23). We showed ha he same also was ue wi h Synechocys is pGln. pGlnCCA was e a ded by M1 RNA bu no pGln (Fig. 5A). No e a ded complex could be obse ed wi h Synechocys is RNase P RNA and ei he pGln o pGlnCCA wi h RNA concen a ions as high as 1 m M, indi- ca ing ha o bo h subs a es he a ini y was oo low o be de ec ed by his assay. The e o e, we ha e de eloped a di e - en assay, in which he p e- RNA was bound o a memb ane and hen incuba ed wi h RNase P RNA. Wi h his assay we could de ec he in e ac ion be ween p e- RNA and Synecho- cys is RNase P RNA (Fig. 5B). We ha e alida ed he assay wi h E. coli M1 RNA. This RNA beha ed in his assay in a way consis en wi h he gel- e a da ion assay. A s onge signal was de ec ed wi h pGlnCCA han wi h pGln, which indica ed ha FIG. 3. E ec o 39 e minal CCA in pGln on p e-s eady-s a e clea age kine ics by RNase P RNA o holoenzyme om E. coli and Synechocys is 6803. Ten picomola 32 P-labeled pGln ( E ) o pGlnCCA ( F ) was incuba ed wi h 20 nM E. coli M1 RNA (A), 40 nM Synecho- cys is RNase P RNA (B), holoenzyme econs i u ed wi h 0.5 nM E. coli M1 RNA and 5 nM C5 p o ein (C), o holoenzyme econs i u ed wi h 2nMSynechocys is RNase P RNA and 20 nM Synechocys is RNase P p o ein (D) and aliquo s wi hd awn a di e en imes. Bu e condi- ions a e as desc ibed in Ma e ials and Me hods. An a e age o h ee independen expe imen s is shown. Table 1. Kine ic cons an s o s eady-s a e clea age o pGln and pGlnCCA by E. coli M1 RNA, Synechocys is RNase P RNA wild ype, and Synechocys is RNase P RNA CCA332 Enzyme Subs a e K m ,M310 6 k ca , min 21 k ca /K m , min 21 zM 21 310 9 E. coli pGln 3.0 60.27 0.039 60.0020 13.0 pGlnCCA 0.13 60.05 0.003 60.0002 23.1 Synechocys is wild ype pGln 2.9 60.25 0.008 60.002 2.8 pGlnCCA 350.6 62.10 0.812 60.012 2.3 Synechocys is CCA332 pGln 2.7 60.45 0.004 60.002 1.5 pGlnCCA 126.5 61.60 0.060 60.005 0.5 K m and k ca we e de e mined as desc ibed in Ma e ials and Me hods. 6674 Biochemis y: Pascual and Vioque P oc. Na l. Acad. Sci. USA 96 (1999) he ela i e in ensi y o he signals was ela ed o he ela i e a ini y be ween bo h RNAs. Synechocys is RNase P RNA, howe e , ga e a clea signal wi h pGln and a ba ely de ec able signal wi h pGlnCCA. The in ensi y o he adioac i e signal was p opo ional o he amoun o p e- RNA used (no shown) and was ep oducible. The e o e, i was possible o compa e he a ini ies o RNase P RNAs o di e en subs a es by quan i ica ion o he adioac i i y bound. Ou esul s ( om iplica e expe imen s simila o he one shown in Fig. 5B) indica ed ha he signal was abou 20- old s onge wi h pGln han wi h pGlnCCA o Synechocys is RNase P RNA, bu i is abou 6- old s onge wi h pGlnCCA han wi h pGln o E. coli M1 RNA. RNA CCA332 beha ed in his assay in a way simila o Synechocys is wild- ype RNase P RNA. DISCUSSION The esul s desc ibed in his pape poin o di e ences in he subs a e- ecogni ion mechanism by cyanobac e ial RNase P as compa ed wi h o he bac e ial RNase P analyzed so a . The 39 e minal CCA in he subs a e, which is an impo an de e minan o subs a e binding in E. coli, has an opposi e e ec in cyanobac e ia. E. coli RNase P RNA o holoenzyme ha e a clea p e e ence o CCA-con aining subs a es. In con as , cyanobac e ial RNase P exhibi s a p e e ence o CCA-lacking subs a es. The di e en ial subs a e p e e ence is due o e ec s bo h on K m and k ca (Table 1). These di e ences esul in an inc eased o e all ca aly ic e iciency (k ca yK m ) o pGlnCCA s. pGln wi h E. coli RNase P RNA o almos 2- old, whe eas he e is a be e ca aly ic e iciency o pGln s. pGlnCCA wi h he Synechocys is RNase P RNA. De ailed kine ic analysis has shown ha he lowe k ca o E. coli wi h CCA-con aining subs a es is due o slow p oduc elease (27, 28). A be e a ini y o he subs a e, as e lec ed in a lowe K m , ansla es in o a g ea e di icul y o eleasing he ma u e RNA p oduc . In con as , Synechocys is RNase P RNA has a much lowe K m o he CCA-lacking subs a e. Tha he subs a e p e e ence is appa en unde single- u no e condi ions, whe e he e is no e ec o p oduc elease, indi- ca es ha he a e o he chemical s ep (k 12 ) also is a ec ed by he p esence o CCA in he subs a e. Mo e de ailed kine ic analysis o he cyanobac e ial sys em is equi ed o con i m his poin . The esul s o he il e -bound RNA–RNA in e ac ion assay a e in ag eemen wi h he kine ic da a. The ela i e in ensi ies o he bands a e cong uen wi h he known RNA–RNA FIG. 4. E ec o 39 e minal CCA in pGln and pGlu on p e-s eady-s a e clea age kine ics by cyanobac e ial RNase P RNAs o holoenzymes. (A–C) Ten picomola p e- RNA (2) o p e- RNACCA (1) was incuba ed wi h he indica ed RNase P RNAs o econs i u ed holoenzymes o 15 min (open ba s) o 30 min (solid ba s), and he amoun o p ocessing was de e mined. Fo he RNA-alone eac ions, he concen a ions used we e 0.5 nM o E. coli M1 RNA and 5 nM o all he o he RNase P RNAs. The E. coli holoenzyme was econs i u ed wi h 0.1 nM M1 RNA and 1 nM C5 p o ein. The Synechocys is wild- ype and CCA332 mu an holoenzymes we e econs i u ed wi h 2 nM RNase P RNA and 20 nM Synechocys is RNase P p o ein. The a e age 6SD o h ee independen expe imen s is shown. (A) pGln and pGlnCCA clea age by RNase P RNA. (B) pGln and pGlnCCA clea age by RNase P holoenzyme. (C) pGlu and pGluCCA clea age by RNase P RNA. (D) Au o adiog am o a ep esen a i e assay gel. Ten picomola 59end-labeled pGln (2) o pGlnCCA (1) was incuba ed o 15 min (lanes 3, 5, 7, 9, 11, and 13) o 30 min (lanes 1, 2, 4, 6, 8, 10, 12, and 14) in he absence o enzyme (lanes 1 and 2) o wi h 0.5 nM E. coli M1 RNA (lanes 3–6), 5 nM Synechocys is RNase P RNA wild ype (lanes 7–10), o 5 nM Synechocys is RNase P RNA CCA332 (lanes 11–14). Biochemis y: Pascual and Vioque P oc. Na l. Acad. Sci. USA 96 (1999) 6675 a ini ies (in E. coli) o as expec ed om he K m alues (Synechocys is): E. coli M1 RNA gi es a s onge signal wi h he CCA-con aining subs a e whe eas Synechocys is RNase P RNA does he opposi e. The di e ences in in ensi ies a e 6- old o E. coli and 20- old o Synechocys is, which is somewha p opo ional o he di e ences in K m (20- old o E. coli and 120- old o Synechocys is). A high di e ence in K m is co ela ed wi h a high di e ence in band in ensi ies in he il e assay. The il e -bound RNA–RNA in e ac ion echnique gene - ally could be use ul o compa ing ela i e RNA–RNA a in- i ies when he a ini y is oo low o be de ec ed by o he adi ional me hods, such as gel e a da ion, and i can be easily scaled up o analyze he binding o many samples wi h a 32 P-labeled RNA o acili a e he compa ison o mu an s, s uc u al a ian s, e c., as well as o he selec ion o s onge - binding RNAs. Why does he Synechocys is enzyme ha e a much lowe a ini y o he CCA-con aining subs a e? The J15y16 loop in E. coli is e y close o he accep o s em o he p e- RNA (29, 30), and i in e ac s wi h he CCA sequence. I is possible ha in Synechocys is he e is s e ic in e e ence when he CCA- con aining subs a e is bound because o he la ge size o he J15y16 loop. Assuming a simila , o e all, h ee-dimensional s uc u e o Synechocys is RNase P RNA and a simila geom- e y o binding o he subs a e o he E. coli sys em, he la ge size o J15y16 would educe he space a ailable o i he subs a e and, he e o e, make i mo e di icul o in e ac wi h CCA-con aining p e- RNAs. A p edic ion o his hypo hesis is ha a educ ion o he size o he J15y16 loop in Synechocys is would educe he p e e ence o CCA-lacking subs a es. The conclusions we ha e eached wi h Synechocys is and pGln can be gene alized o o he subs a es, such as pGlu, and o o he cyanobac e ia, such as Anabaena 7120 and Pseudan- abaena 6903. The e a e published epo s ha he RNase P RNA om he cyanobac e ia P ochlo o h ix hollandica and P ochlo ococcus ma inus a e s ongly dependen on he p es- ence o CCA in he subs a e o ac i i y (31, 32), in con a- dic ion wi h ou esul s. In hose s udies, a plan chlo oplas subs a e was used whe eas we ha e used a comple ely ho- mologous sys em. The e o e, i would be in e es ing o con i m wi h a cyanobac e ial p e- RNA whe he P. hollandica and P. ma inus a e ue excep ions o ou obse a ion ha cyanobac- e ia ha e a p e e ence o CCA-lacking subs a es. The J15y16 loop is impo an in E. coli no only o subs a e binding bu also o ca alysis. Single base subs i u ions on his loop ha e s ong e ec s on he kine ic cons an s and on he clea age-si e selec ion (11, 28, 33, 34). Al hough a signi ican change (i.e., a 3-n dele ion) in he J15y16 loop was in oduced in he Synechocys is RNase P, he e ec o he change was minimal on K m and i was signi ican on k ca only wi h he CCA-con aining subs a e. The change had no signi ican e ec on he subs a e p e e ence o he enzyme, and i did no a ec clea age-si e selec ion wi h ei he subs a e. This, again, sugges s ha he ole o J15y16 in ca alysis by cyanobac e ial RNase P is no as ele an as wi h E. coli o o he bac e ial RNase P. The 39 e minal CCA in e ac ion wi h he J15y16 loop is used in E. coli as pa o a measu ing de ice o de e mine he clea age si e (34–37). P esumably, in cyanobac- e ia, as in euka yo es, whe e he CCA also is unimpo an , al e na i e con ac s a e used o de e mine he clea age si e (2). Ou esul s would explain he la ge a iabili y in sequence and s uc u e o J15y16 in cyanobac e ia compa ed wi h o he bac e ia (14). When he e a e no unc ional cons ain s on RNA s uc u e, i will e ol e mo e eely and explo e in a neu al way a la ge ange o possible s uc u es. RNA genes in cyanobac e ia do no code o he 39 e minal CCA, and, he e o e, i is possible ha cyanobac e ial RNase P unc ion has e ol ed in a con ex in which he CCA sequence is no p esen . In some cases, an ex a helix has been inse ed in his egion (14, 31). The inse ion, dele ion, o subs i u ion o s uc u es du ing he e olu ion o RNase P RNA is wide- sp ead. They e lec ei he speci ic adap a ions in he di e en RNase P RNAs o andom changes in unc ionally neu al si es. A good example is p esen ed by cyanobac e ia again: in he e ocys - o ming s ains, helix P12 is much la ge han usual and con ains a sho , andemly epea ed sequence (14). These epea s seem o ha e been inse ed ecen ly and independen ly du ing he e olu ion o RNase P RNA in hese s ains. In e es ingly, hey can be dele ed wi hou signi ican e ec on he kine ics o clea age (14) o on he binding o he p o ein subuni (unpublished da a). In summa y, ou esul s indica e ha cyanobac e ial RNase P,in con as oo he bac e ia,doesno equi e he39 e minal CCA in he subs a e o e icien ac i i y. CCA is de imen al o ac i i y and educes he a ini y be ween enzyme and subs a e. This esul is in ag eemen wi h he ac ha CCA is no encoded in cyanobac e ial RNA genes bu is added pos ansc ip ionally, which suppo s he idea ha RNase P e ol es o adap o he p ima y sequence o RNAs as p o- posed ecen ly (16). 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