Recombinant bacterial RNA polymerase: Preparation and applications

Methods 47 (2009) 44–52

Contents lists available at ScienceDirect

Methods j o u r n a l h o m e p a g e : w w w . e l s e v i e r. c o m / l o c a t e / y m e t h

Recombinant bacterial RNA polymerase: Preparation and applications Konstantin Kuznedelov a,*, Konstantin Severinov a,b,c a

Waks­man Insti­tute, Rut­gers, The State Uni­ver­sity of New Jer­sey, 190 Fre­linghuy­sen Road, Pis­cat­a­way, NJ 08854, USA Insti­tute of Molec­u­lar Genet­ics, Mos­cow, Russian Federation c Insti­tute of Gene Biol­ogy, Rus­sian Acad­emy of Sci­ences, Mos­cow, Russian Federation b

a r t i c l e

i n f o

Article history: Accepted 8 October 2008 Available online 21 October 2008  Key­words: RNA poly­mer­ase Tran­scrip­tion Bac­te­rial In vitro recon­sti­tu­tion Co-over­ex­pres­sion Aquifex ae­o­li­cus

a b s t r a c t Avail­abil­ity of DNA-depen­dent RNA poly­mer­ase from var­io ­ us bac­te­ria is a key for set­ting up spe­cific in vitro tran­scrip­tion sys­tems nec­es­sary for under­stand­ing spe­cies-spe­cific tran­scrip­tion reg­u­la­tion. We describe here two main strat­e­gies for recombinant RNA poly­mer­ase prep­a­ra­tion—through in vitro recon­sti­tu­tion and het­er­ ol­o­gous co-over­pro­duc­tion in Esch­e­richia coli. Both strat­e­gies can be used for prep­a­ra­tion of large amounts of RNA poly­mer­ases from any bac­te­ria for which sequences of rpo (RNA poly­mer­ase) genes are known. © 2008 Elsevier Inc. All rights reserved.

1. Intro­duc­tion DNA-depen­dent RNA poly­mer­ase (RNAP) is the key enzyme of gene expres­sion. RNAP is a mul­ti­sub­unit, mul­ti­func­tional molec­ u­lar machine, whose func­tions are reg­u­lated by var­i­ous cel­lu­ lar fac­tors in response to envi­ron­men­tal cues. Despite the very high degree of evo­lu­tion­ary con­ser­va­tion at and around the cat­ a­lytic cen­ter, RNAP from dif­fer­ent organ­isms, includ­ing dif­fer­ent ­eu­bac­te­ria, exhibit spe­cies-spe­cific prop­er­ties such as spec­i­fic­ity of pro­moter rec­og­ni­tion or abil­ity to inter­act with and respond to tran­scrip­tion fac­tors. Thus, under­stand­ing tran­scrip­tion reg­u­la­tion in a par­tic­u­lar bac­te­rium often means that an in vitro tran­scrip­tion sys­tem with cog­nate RNAP needs to be set up. Bac­te­rial RNAP con­sists of a cat­al­ yt­i­cally pro­fi­cient core enzyme (sub­unit com­po­si­tion a2bb9x, molec­u­lar weight »300–400 kDa) and a spec­i­fic­ity sub­unit r. A com­plex of core with r is called the holo­en­zyme and is able to spe­cif­i­cally rec­og­nize pro­mot­ers. Dif­ fer­ent r fac­tors direct the core to dif­fer­ent groups of pro­mot­ers. The small­est RNAP sub­unit, x (»10 kDa) is the only sub­unit that is dis­pens­able for most in vivo and in vitro func­tions of the enzyme. While robust pro­ce­dures suit­able for RNAP puri­fi­ca­tion from most bac­te­ria have been devel­oped, their appli­ca­tion is ham­pered by dif ­fi­cul­ties asso­ci­ated with cul­ti­vat­ing, let alone grow­ing large vol­ umes of bac­te­rial cul­tures needed for bio­chem­i­cal frac­tion­ation. Bac­te­rial RNAP is unique in that it can be effi­ciently recon­sti­tuted in vitro from iso­lated recombinant sub­units, or prepared by het­er­ ol­og ­ ous co-over­ex­pres­sion of sub­units in sur­ro­gate hosts, open­ing way for func­tional and struc­tural anal­y­ses of tran­scrip­tion mech­a­ * Cor­re­spond­ing author. Fax: +1 732 445 5735. E-mail address: kuz­ned­el­[email protected]­man.rut­gers.edu (K. Kuznedelov). 1046-2023/$ - see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.ymeth.2008.10.007

nism and reg­u­la­tion in hard-to-cul­ti­vate micro­or­gan­isms. In addi­ tion, both the in vitro assem­bly and co-over­ex­pres­sion approaches allow pre­par­ing RNAP mutants lack­ing essen­tial func­tions. This is very impor­tant for reverse genet­ics anal­y­sis of RNAP, which is an essen­tial enzyme, mean­ing that clas­si­cal genetic approaches are lim­ited to anal­y­sis of via­ble muta­tions. Below, we pres­ent detailed pro­to­cols for prep­a­ra­tion of recombinant RNAP by in vitro assem­ bly and co-over­ex­pres­sion. 2. In vitro recon­sti­tu­tion of recombinant bac­te­rial RNAP A gen­eral scheme of in vitro recon­sti­tu­tion of bac­te­rial RNAP is pre­sented in Fig. 1 and is described in detail in the fol­low­ing sec­ tions. 2.1. Expres­sion of recombinant RNAP sub­units in Esch­e­richia coli Plas­mids of the pET series (Nova­gen) have been suc­cess­fully used as vec­tors for expres­sion of rpo genes from var­i­ous bac­te­rial sources. Plas­mids with rpo genes are trans­formed into the E. coli BL21(DE3) cells and trans­for­mants are plated on solid medium con­tain­ing appro­pri­ate anti­bi­ot­ics. In PET-based plas­mids, the tar­get pro­tein over­pro­duc­tion is induced by the addi­tion of IPTG (usu­ally 1 mM). It is very impor­tant to find opti­mal con­di­tions for over­pro­duc­tion for each RNAP sub­unit. In our expe­ri­ence, vary­ ing growth tem­per­a­ture, IPTG con­cen­tra­tion, cul­ture OD600 at the time of induc­tion, and cul­ti­va­tion time after the induc­tion allows­ high- to medium lev­els of expres­sion of RNAP sub­units from var­i­ous sources, includ­ing Gram-neg­a­tive bac­te­ria (E. coli, Xan­ tho­mo­nas ory­zae, and Fran­ci­sel­la tu­lar­en­sis), Gram-positive bac­ te­ria (Bacil­lus sub­til­is, Bacil­lus cereus), and sev­eral ther­mo­philic



K. Ku­zn­ede­lov, K. Se­ver­i­nov / Methods 47 (2009) 44–52

45

should be pre­heated to 37 oC. The cul­ture is grown at 37 °C with vig­or­ous agi­ta­tion until OD600 reaches 0.4–0.8 (usu­ally 2–3 h), induced with 1 mM IPTG and allowed to grow fur­ther (usu­ally for addi­tional 2–5 h; the opti­mal time of induc­tion time could be deter­mined dur­ing the pilot exper­i­ments). A 0.1–0.5 ml ali­quot of the cul­ture is with­drawn before the induc­tion and stored at room tem­per­a­ture in an Ep­pen­dorf tube. When the induc­tion is com­ plete, another ali­quot of the cul­ture is with­drawn. Cells from both cul­ture ali­quots are col­lected by cen­tri­fu­ga­tion in a micro­cen­tri­ fuge, the super­na­tant is removed, and the pellet is resus­pended in 20 ll H2O and equal vol­ume of Lae­mmli load­ing buffer is added. Sam­ples are boiled for 5 min and ana­lyzed by SDS–PAGE to deter­ mine recombinant RNAP sub­units expres­sion lev­els. If desired lev­ els of over­pro­duc­tion are detected, cells from induced cul­tures are col­lected by cen­tri­fu­ga­tion (4000g, 10 min, 4 °C), the medium is removed and cell pellet drained and stored at ¡80 °C until fur­ther use. 2.2. Inclu­sion bodies prep­a­ra­tion and puri­fic­ a­tion of His-tagged RNAP sub­units from cell extracts Esch­er­ ichia coli BL21(DE3) cells trans­formed with rpo gene expres­sion plas­mids usu­ally over­pro­duce indi­vid­ual core RNAP sub­units at high-level upon induc­tion and these RNAP sub­units often form inclu­sion bodies. Nev­er­the­less, it is very impor­tant to deter­mine the local­i­za­tion (cyto­plas­mic ver­sus inclu­sion bodies) of recombinant pro­tein when over­pro­duc­ing an RNAP sub­unit from a new source. This is best done by performing a small-scale trial induc­tion in »10 ml of LB medium. It is safer to per­form local­ i­za­tion tri­als by grow­ing cell cul­tures in tiny flasks rather than tubes, since induc­tion con­di­tions often do not scale prop­erly from tubes to flasks. Induced cells are col­lected in an Ep­pen­dorf tube, resus­pended in 400–500 ll of lysis buffer (see Table 1) and lysed by sev­eral 5- to 10-s son­i­ca­tion bursts using a mi­cro­tip with 1-min rests between the bursts. The tube shall be kept in a water-ice bath dur­ing the son­i­ca­tion. An ali­quot of lysed cells is next removed and cell debris and inclu­sion bodies are col­lected by a 2–5 min cen­tri­ fu­ga­tion in a refrig­er­ated micro­cen­tri­fuge. An ali­quot of the cleared cell lysate is removed, the rest of the super­na­tant dis­carded and the pellet is resus­pended in an ini­tial vol­ume of the lysis buffer

Table 1 Solu­tions and buf­fers. Fig. 1. Prep­a­ra­tion of bac­te­rial RNAP core enzyme by in vitro recon­sti­tu­tion. The sequence of steps involved in RNAP core enzyme recon­sti­tu­tion is sche­mat­i­cally pre­sented. The x sub­unit can be omit­ted from recon­sti­tu­tion reac­tion with min­i­mal effects on most RNAP func­tions. See text for details.

­ rgan­isms (Ther­mus aquat­i­cus and Aquifex ae­o­li­cus). Should prob­ o lems with expres­sion be encoun­tered, var­i­ous pro­ce­dures such as alter­ing the ratio of iso­ac­cept­ing tRNAs in the expres­sion host can be used to attempt to increase the yield. Use­ful infor­ma­tion about ­opti­mi­za­tion of induc­tion can be found in the PET sys­tem man­ual from Nova­gen (http://www.emd­bio­scienc­es.com/docs/docs/PROT/ TB055.pdf). Once the induc­tion con­di­tions have been opti­mized in pilot exper­i­ments, large-scale induc­tion is per­formed. Only freshly trans­formed cells (grown at 37 oC for no more than 12 h) should be used. Cells are col­lected from plates by scrap­ing with a micro­ bi­o­log­i­cal loop and are care­fully resus­pended in »1 ml of liquid LB medium in a ster­ile Ep­pen­dorf tube. The result­ing sus­pen­sion is used to inoc­u­late liquid cul­tures (a plate con­tain­ing sev­eral hun­dred »1 mm col­o­nies is suf ­fi­cient for inoc­u­la­tion of 1 l of LB medium). The medium should con­tain appro­pri­ate anti­bi­ot­ics and

Solu­tion name

Com­po­nents

Com­ments

Lysis buffer

40 mM Tris–HCl, pH 7.9; 300 mM KCl; 10 mM EDTA

Grind­ing buffer

40 mM Tris–HCl, pH 7.9; 100 mM NaCl; 10 mM EDTA

Stor­age buffer

40 mM Tris–HCl, pH 7.9; 200 mM KCl; 50% glyc­erol; 1 mM EDTA; 1 mM 2-ME 6 M gua­ni­dine–HCl; 50 mM Tris–HCl, pH 7.9; 10 mM MgCl2; 10 lM ZnCl2; 10% glyc­erol; 1 mM EDTA 50 mM Tris–HCl pH 7.9; 200 mM KCl; 10 mM MgCl2; 10 lM ZnCl2; 10% glyc­erol; 1 mM EDTA 40 mM Tris–HCl, pH 7.9; 5% glyc­erol; 1 mM EDTA 20 mM Tris–HCl, pH 7.9; (or Hepes); 500 mM NaCl; 5% glyc­erol 40 mM Tris–HCl, pH 8.4; 40 mM KCl; 10 mM MgCl2

Before use sup­ple­mented with 15 mM 2-ME and 0.1 mM PMSF Before use sup­ple­mented with 15 mM 2-ME and 0.1 mM PMSF Used for dial­y­sis

Dena­tur­ation buffer

Recon­sti­tu­tion buffer

TGE buffer Start buffer

Tran­scrip­tion buffer

Before use sup­ple­mented with 10 mM DTT

Before use sup­ple­mented with 1 mM DTT or 2-ME (see text) Before use sup­ple­mented with 1 mM 2-ME For metal che­late affin­ity chro­ma­tog­ra­phy

46

K. Ku­zn­ede­lov, K. Se­ver­i­nov / Methods 47 (2009) 44–52

with a brief burst of son­i­ca­tion. An ali­quot of the result­ing homo­ ge­neous sus­pen­sion is removed. The three ali­quots (crude lysate, cleared lysate, and pellet sus­pen­sion) are next ana­lyzed by SDS– PAGE and the amount of the tar­get pro­tein in sol­u­ble and insol­u­ble frac­tions is esti­mated. Incom­plete cell lysis can lead to errors in esti­mates of insol­u­ble pro­tein. The eas­i­est way to deter­mine that lysis is com­plete is to mon­i­tor the pres­ence of the E. coli b and b9 RNAP sub­unit bands in the inclu­sion bodies frac­tion. These two sub­units form very char­ac­ter­is­tic closely spaced bands with appar­ ent elec­tro­pho­retic mobil­i­ties of 150 and 155 kDa. They shall be absent in the inclu­sion bodies sam­ple (obvi­ously, this method is not appli­ca­ble when an over­ex­pres­sed sub­unit matches the size of E. coli b and or b9). The fol­low­ing pro­ce­dure can be used for large-scale prep­a­ra­ tion of RNAP sub­unit con­tain­ing lysates or inclu­sion bodies. Cells (fro­zen or freshly col­lected) are resus­pended in lysis buffer with 15 mM b-mercap­toethanol (2-ME) and 0.1 mM PMSF (5 ml of buffer per 1 g of bac­te­rial paste). A homo­ge­neous cell sus­pen­sion is prepared using a glass rod or by vig­or­ous pipet­ting through a glass or plas­tic pipette. The cell sus­pen­sion is trans­ferred into a glass bea­ker or a cen­tri­fuge tube. Cells are dis­rupted by 1-min bursts of son­i­ca­tion using max­i­mal power set­ting for a tip used (var­ies depend­ing on the total vol­ume of cell sus­pen­sion) at a 50% duty cycle. Dur­ing son­i­ca­tion, the ves­sel with cell sus­pen­sion is kept in a water-ice bath. The sus­pen­sion is left for 1 min in a water-ice bath to cool down, and then the son­ic­ a­tion step is repeated (4–5 times with 1 min rests between each son­i­ca­tion give sat­is­fac­tory results). The lysate is next cen­tri­fuged at 18,000g for 1 h at 4 °C. Such long cen­tri­fu­ga­tion step is only war­ranted if an over­pro­duced RNAP sub­unit is found to be sol­u­ble. A 5- to 10-min cen­tri­fu­ga­tion is suf ­fi­cient to pellet inclu­sion bodies. The super­na­tant is either dis­carded (if over­pro­duced pro­tein is mostly located in inclu­sion bodies) or, if it con­tains over­pro­duced pro­tein, trans­ferred into a 50 ml screw-cap poly­pro­pyl­ene tube. To the super­na­tant, dry finely grained (NH4)2SO4 is added to 60% sat­u­ra­tion and dis­solved com­ pletely by gen­tle mix­ing. The tube is left on ice for at least 1 h for pro­tein pellet to form. Pro­teins pre­cip­i­tated with (NH4)2SO4 can be stored at +4 °C indef­i­nitely (in the form of sus­pen­sion in (NH4)2SO4con­tain­ing mother solu­tion) until fur­ther use. A pellet con­tain­ing inclu­sion bodies is fur­ther washed by repeated cycles of son­i­ca­tion (as described above) and cen­tri­fu­ga­ tion, first in the lysis buffer, then in the same buffer con­tain­ing 0.2% Na–deoxy­cho­late. The final pellet is resus­pended in a small vol­ume of lysis buffer (2 ml of buffer per 1 g of start­ing bac­te­rial paste) and ali­quots of 0.2 ml are placed in indi­vid­ual Ep­pen­dorf tubes. The tubes are cen­tri­fuged for 5 min in a micro­cen­tri­fuge, super­na­tants are dis­carded and pel­lets are stored at ¡80 °C until use. Puri­fi­ca­tion of sol­ub ­ le untagged RNAP sub­units from cell extracts can be com­pli­cated since at least large sub­units (b and b9) are not prop­erly folded and do not behave well dur­ing chro­ mato­graphic steps. There­fore, if over­pro­duced sub­unit is found sol­u­ble, it is best to affin­ity tag it by genet­i­cally fus­ing to a ter­mi­ nal hexa­his­ti­dine tag and purify it by metal che­late affin­ity chro­ ma­tog­ra­phy. In our expe­ri­ence, affin­ity tags at either ter­mi­nus of RNAP sub­units from var­i­ous sources have min­i­mal effects on most RNAP func­tions. If a tag is of con­cern, it can be removed by treat­ ment with spe­cific pro­te­ases, since most PET vec­tors incor­po­rate a pro­te­ase rec­og­ni­tion site between the affin­ity puri­fi­ca­tion tag and the tar­get pro­tein, Col­lect the (NH4)2SO4 pre­cip­i­tate (see above) by 20-min cen­ tri­fu­ga­tion at 15,000g, 4 °C. Care­fully dis­card the super­na­tant and drain the pellet by plac­ing the cen­tri­fuge tube upside down on a paper towel. Wipe the remain­ing drops of the super­na­tant from the cen­tri­fuge tube walls using Ki­mw­i­pes (avoid touch­ing the pellet). Dis­solve pellet in 5 ml of start buffer (Table 1) con­tain­ ing 5 mM imid­az­ole (avoid cre­at­ing bub­bles when dis­solv­ing the

pellet). For bet­ter bind­ing of the tar­get pro­tein, it is some­times worth using Hepes instead of Tris–HCl (Tris–HCl tends to reduce pro­tein bind­ing by metal affin­ity col­umns). Remove undis­solved mate­rial by 30-min cen­tri­fu­ga­tion at 15,000g, 4 °C. Trans­fer the super­na­tant into a fresh screw-cap poly­pro­pyl­ene tube and repeat the cen­tri­fu­ga­tion step. Fil­ter the super­na­tant through a 0.45 lm Nylon Mem­brane acrylic fil­ter (PALL, Life Sci­ences) attached to a syringe and load onto a 1 ml Hi-Trap che­lat­ing col­umn (GE Health­ care) or equiv­a­lent charged with Ni2+ accord­ing to man­u­fac­turer’s instruc­tions and equil­i­brated in the start buffer. The col­umn can be either attached to a liquid chro­ma­tog­ra­phy sys­tem or (an eas­ier and faster way) all steps can be per­formed man­u­ally with a syringe. Wash the col­umn with 5 ml of start buffer with 20 mM imid­az­ole (up to 60 mM of imid­az­ole can be used at the wash step; the con­ cen­tra­tion of imid­az­ole at the wash step should be deter­mined exper­i­men­tally for each RNAP sub­unit: for exam­ple RNAP a sub­ unit tends to bind to the col­umn stron­ger because of the biden­tate nature of the inter­ac­tion—since a is a dimer at native con­di­tions). Because of con­sid­er­able var­i­abil­ity between metal ion che­lat­ing res­ins from dif­fer­ent sources and even between batches from the same source, opti­mal bind­ing and elu­tion con­di­tions should be adjusted each time a new col­umn is used. In gen­eral, the higher the con­cen­tra­tion of imid­az­ole at the wash step the purer the final prep­a­ra­tion of the pro­tein. Elute the pro­tein with start buffer con­tain­ing 100 mM imid­az­ole col­lect­ing 1 ml frac­tions (some­times, up to 500 mM imid­az­ole is required for com­plete elu­tion, this should be deter­mined exper­i­men­tally). Ana­lyze all frac­tions by SDS–PAGE and pool those con­tain­ing the tar­get sub­unit, which should be 70–90% pure. Con­cen­trate pooled ­frac­tions by ultra­fil­tra­tion using a Mi­cro­sep (PALL, Life Sci­ence) cen­trif­u­gal device (or an anal­o­gous one) with the appro­pri­ate molec­u­lar weight cut-off to a final pro­tein con­cen­tra­tion of no less than 1 mg/ml, dia­lyze against stor­age buffer (see Table 1) and store at ¡20 °C. Note that dial­y­sis in 50% glyc­erol buffer fur­ther con­cen­ trates the pro­tein. It is some­times nec­es­sary to purify His-tagged RNAP sub­units at dena­tur­ing con­di­tions (for exam­ple, to decrease the back­ground of E. coli RNAP sub­units that may asso­ci­ate with het­er­ol­o­gously over­ pro­duced RNAP sub­unit). Puri­fi­ca­tion is car­ried out the same way as described above but 6 M gua­ni­dine–HCl is added to all buf­fers. Frac­tions con­tain­ing pure pro­tein are pooled and dia­lyzed against recon­sti­tu­tion buffer (see Table 1) with 5 mM 2-ME. Pre­cip­i­tate formed dur­ing the dial­y­sis is removed by 30-min cen­tri­fu­ga­tion at 15,000g, 4 °C, sol­u­ble pro­tein is con­cen­trated and stored as above. 2.3. RNAP recon­sti­tu­tion from indi­vid­ual sub­units RNAP recon­sti­tu­tion pro­ce­dure devel­oped for E. coli RNAP and described in detail else­where [1] can be suc­cess­fully used for recon­sti­tu­tion of RNAP from other bac­te­rial spe­cies. Below, we pres­ent a recon­sti­tu­tion pro­to­col based on this pro­ce­dure with some mod­i­fi­ca­tions. Prior to in vitro recon­sti­tu­tion, inclu­sion bodies are sol­u­bi­lized in dena­tur­ation buffer (Table 1) with 10 mM DTT (added imme­ di­ately before use). For more effi­cient sol­u­bi­li­za­tion, the inclu­ sion bodies pellet from a sin­gle ali­quot (above) is resus­pended by pipet­ting and the sus­pen­sion is left for 30 min on ice. Undis­solved mate­rial is removed by 30-min cen­tri­fu­ga­tion in a micro­cen­tri­fuge (4 °C). The super­na­tant is trans­ferred into a fresh tube and pro­tein con­cen­tra­tion is deter­mined using the Brad­ford assay with BSA as a stan­dard. RNAP sub­units dete­ri­o­rate after sol­u­bi­li­za­tion in the dena­ tur­ation buffer and should be used shortly after sol­u­bi­li­za­tion. RNAP sub­units are mixed in a molar ratio of 2:8:4 (a:b:b9), the total pro­tein con­cen­tra­tion is adjusted to 0.5 mg/ml with dena­ tur­ation buffer con­tain­ing 10 mM DTT and the mix­ture is dia­lyzed



K. Ku­zn­ede­lov, K. Se­ver­i­nov / Methods 47 (2009) 44–52

(at 4 °C) for 16 h against two changes of 250 vol­umes of recon­sti­ tu­tion buffer con­tain­ing 10 mM 2-ME. Any pre­cip­i­tate formed dur­ ing dial­y­sis is removed by cen­tri­fu­ga­tion. To the super­na­tant, one molar equiv­a­lent of the primary RNAP r sub­unit in stor­age buffer is added and the mix­ture is incu­bated for 1 h at 30 °C. This “ther­ mo­ac­ti­va­tion” step allows the holo­en­zyme to form and seems to gen­er­ally increase the yield of assem­bled active enzyme. The addi­ tion of r step can be omit­ted, espe­cially if RNAP core is prepared. How­ever, total yields (and spe­cific activ­ity of resul­tant RNAP) become con­sid­er­ably lower, The result­ing RNAP prep­a­ra­tions can be used directly in tran­ scrip­tion assays or stored under (NH4)2SO4 (65% sat­u­ra­tion) until fur­ther use. RNAP in recon­sti­tu­tion mix­tures is not very sta­ble and loses activ­ity with time, even if stored in the pres­ence of 50% glyc­ erol at ¡20 °C. Sta­ble pure RNAP can be obtained by subsequent Su­pe­rose-6 gel-fil­tra­tion and Mono-Q or Resource-Q ion-exchange chro­mato­graphic puri­fi­ca­tion steps. In vitro recon­sti­tu­tion con­di­tions described above always yield a mix­ture of RNAP sub­as­sem­blies and core in dif­fer­ent ratios; some­ times, espe­cially when work­ing with RNAP mutants, no assem­bled enzyme is pro­duced (see, for exam­ple, Ref. [9]). The Su­pe­rose-6 step sep­a­rates assem­bled enzyme from assem­bly inter­me­di­ates and unas­sem­bled sub­units and allows one to deter­mine whether assem­bly of recombinant RNAP occurred in the first place, judg­ing by the appear­ance of char­ac­ter­is­tic chro­mato­graphic peaks. The pellet obtained after (NH4)2SO4 pre­cip­i­ta­tion of RNAP recon­sti­tu­ tion mix­ture is col­lected by cen­tri­fu­ga­tion, drained thor­oughly and dis­solved in 0.25 ml of TGE buffer (see Table 1) with 1 mM 2-ME. Undis­solved mate­rial is removed by sev­eral cen­tri­fu­ga­tion steps (until no vis­i­ble pellet is formed) and the super­na­tant is loaded on a Su­pe­rose-6 10/30 HR (GE Health­care) col­umn attached to an FPLC and equil­i­brated in the TGE buffer con­tain­ing 200 mM NaCl and 1 mM 2-ME. The chro­ma­tog­ra­phy is con­ducted in the same buffer (0.4 ml/min flow rate); 1 ml frac­tions are col­lected and ana­lyzed by SDS–PAGE. Frac­tions con­tain­ing RNAP (and the a2b sub­as­sem­bly) usu­ally form a sharp peak that elutes after a peak of aggre­gates (elutes in void vol­ume) and a dif­fuse peak of unas­sem­bled large RNAP sub­units. The Mono-Q step sep­a­rates the a2b sub­as­sem­bly, RNAP core, and holo­en­zyme from each other and also removes excess r and con­ tam­i­nat­ing ribo­nu­cle­ases. Su­pe­rose-6 frac­tions con­tain­ing RNAP are pooled, diluted 2-fold with TGE buffer, and loaded (1 ml/min flow rate) onto a 1 ml Mono-Q col­umn equil­i­brated with TGE con­ tain­ing 100 mM NaCl and attached to FPLC. The col­umn is washed with 10 ml of the same buffer and bound pro­teins are eluted with a lin­ear gra­di­ent of NaCl (from 200 to 400 mM NaCl) in TGE over the course of 35 min. The col­umn is next washed with TGE con­tain­ ing 1 M NaCl. Dur­ing Mono-Q chro­ma­tog­ra­phy, RNAP core and the holo­en­zyme elute sep­a­rately (RNAP core first, in a series of closely spaced and poorly sep­ar­ ated peaks that prob­ab ­ ly rep­re­sent dif­ fer­ent con­for­ma­tion of the core, fol­lowed by a sharp holo­en­zyme peak). The r sub­unit elutes last. RNAP core and holo­en­zyme con­ tain­ing frac­tions are iden­ti­fied by SDS–PAGE, pooled, made 50% with glyc­erol and stored at ¡20 °C. Alter­na­tively, RNAP in pooled frac­tions can be con­cen­trated »4-fold by dia­lyz­ing against the stor­age buffer. If the amount of RNAP is low, frac­tions can be con­ cen­trated on a cen­trif­u­gal device such as Mi­cro­sep 100 K Omega (PALL, Life Sci­ence) fol­lowed by the addi­tion of glyc­erol to 50%. The con­cen­tra­tion of RNAP is deter­mined using the Brad­ford assay with BSA as a stan­dard or, pref­er­a­bly, deter­mined spec­tro­pho­to­ met­ri­cally using the cal­cu­lated extinc­tion coef ­fi­cient at 280 nm. At con­cen­tra­tions of 1 mg/ml or more, thus puri­fied RNAP sam­ples remain active for many years when stored at ¡20 °C. If any RNAP sub­unit is affin­ity tagged, the enzyme can be batchpuri­fied by metal che­late affin­ity chro­ma­tog­ra­phy [2]. To this end, recon­sti­tu­tion mix­tures after dial­y­sis and ther­mo­ac­ti­va­tion (above)

47

are com­bined with the appro­pri­ate amount Ni2+–NTA aga­rose (Qiagen) equil­i­brated with 50 mM Tris–HCl pH 7.9, 0.5 mM EDTA, 5% glyc­erol. After »30 min bind­ing with gen­tle agi­ta­tion, aga­rose beads are washed three times with the same buffer con­tain­ing 5 mM imid­az­ole, and bound pro­tein is eluted with the buffer con­ tain­ing 150 mM imid­az­ole. The sam­ples are con­cen­trated (and the buffer changed to one with­out imid­az­ole) using a cen­trif­ug ­ al ultra­ fil­tra­tion device and the pro­tein is stored as above. Thus puri­fied RNAP is suit­able for most in vitro tran­scrip­tion assays but tends to dete­ri­o­rate upon long-term stor­age (stor­ing under (NH4)2SO4 helps to avoid this prob­lem). 3. Prep­a­ra­tion of recombinant RNAP by co-over­ex­pres­sion in E. coli The first suc­cess­ful prep­a­ra­tion of func­tional RNAP by co-expres­ sion in E. coli of a het­er­ol­o­gous set of rpo genes was achieved for T. aquat­i­cus RNAP [3], the first RNAP for which a high-res­o­lu­tion struc­ture became avail­able [4]. This exper­i­men­tal sys­tem was later improved and used for prep­a­ra­tion of mil­li­gram amounts of sev­ eral struc­ture-based recombinant T. aquat­i­cus RNAP mutants [5–8]. Since then, enzymes from sev­eral other sources were prepared by this method. Of note here is the prep­a­ra­tion of recombinant E. coli RNAP har­bor­ing a large dele­tion in an evo­lu­tion­arily var­i­able region of the b’ sub­unit [9] and the prep­a­ra­tion of crys­tal­liz­able recombinant T. aquat­i­cus RNAP [7]. In the former case, the mutant enzyme could not have been prepared by in vitro recon­sti­tu­tion, pos­si­bly reflect­ing the role of pro­tein chap­er­ones in the pro­cess of RNAP assem­bly in vivo. In the lat­ter exam­ple, co-over­ex­pres­ sion turned out to be the only way to obtain recombinant RNAP form­ing dif­frac­tion qual­ity crys­tals. Again, the supe­rior qual­i­ties of recombinant enzyme obtained by co-over­ex­pres­sion (as com­ pared to one prepared by in vitro recon­sti­tu­tion) may either reflect the pres­ence of chap­er­ones (which, how­ever, must be spe­cies nonspe­cific) or result from spe­cial con­di­tions of in vivo assem­bly such as co-trans­la­tional fold­ing, ionic con­di­tions etc. For every RNAP obtained by co-over­ex­pres­sion, a rather labo­ri­ ous pro­ce­dure of cre­at­ing a large PET-based plas­mid with rpo genes expressed from indi­vid­ual T7 RNAP pro­mot­ers or jointly expressed from a sin­gle pro­moter as part of an arti­fi­cial operon needs to be accom­plished. The par­tic­u­lar strat­egy depends very much on the sequence and avail­abil­ity of restric­tion sites in rpo genes of a micro­or­gan­ism under study. In gen­eral, PET-based plas­mids used for indi­vid­ual expres­sion of rpo genes for in vitro recon­sti­tu­tion serve as a use­ful point of depar­ture. Var­i­ous arrange­ments of rpo genes in the final co-over­ex­pres­sion plas­mids have led to suc­cess­ ful prep­a­ra­tion of recombinant RNAP, sug­gest­ing that gene order is not par­tic­u­larly impor­tant. Since co-over­ex­pres­sion strat­egy for Ther­mus RNAP is ade­quately described in pub­lished lit­er­a­ture [3,6,7], below, we pres­ent the appli­ca­tion of the same strat­egy to obtain and ini­tially char­ac­ter­ize RNAP from hy­per­therm­o­phil­ic Gram-neg­a­tive eubac­te­rium A. ae­o­li­cus. Cur­rently, co-over­ex­pres­ sion strat­egy is most suc­cess­ful when pre­par­ing RNAP from ther­ mo­philic organ­isms (or more gen­er­ally, RNAPs that sub­stan­tially dif­fer from E. coli RNAP in terms of their phys­i­cal sta­bil­ity), since clean sep­a­ra­tion of the tar­get enzyme from con­tam­i­nat­ing E. coli RNAP and, more impor­tantly, from inter­spe­cies hybrid enzymes becomes pos­si­ble. Esch­er­ ichia coli mutants defec­tive in RNAP assem­bly due to muta­tions in all genes cod­ing for RNAP core sub­ units (except for x) have been described [10,11]. It is likely that the use of such mutant strains or their deriv­a­tives as hosts for co-over­ ex­pres­sion will allow the use of co-over­ex­pres­sion strat­egy for prep­a­ra­tion of micro­bial RNAPs whose phys­i­cal prop­er­ties resem­ ble those of the E. coli RNAP. Use of multiple orthog­o­nal affin­ity tags posi­tioned on dif­fer­ent recombinant RNAP sub­units should also be help­ful to decrease con­tam­i­na­tion by hybrid enzymes.

48

K. Ku­zn­ede­lov, K. Se­ver­i­nov / Methods 47 (2009) 44–52

3.1. Clon­ing Aquifex ae­ol­i­cus rpo genes in E. coli expres­sion and co-expres­sion plas­mids Prim­ers for PCR ampli­fi­ca­tion of A. ae­o­li­cus rpoA, rpoB, rpoC, rpoZ, and rpoD genes were designed using avail­able A. ae­o­li­cus genome sequence data [12]. The prim­ers allowed the clon­ing of each of ampli­fied A. ae­o­li­cus rpo genes in pET series plas­mids between the NdeI (or NcoI) and Eco­RI (or Bam­HI or NotI) sites of the pol­y­linker. Plas­mids pET11-AaeA, pET28-AaeB, pET28-AaeC, pET28-AaeZ, and pET11-AaeD and pET28-AaeD over­express­ing, respec­tively, untagged A. ae­o­li­cus RNAP a and b sub­units, C-ter­ mi­nally hexa­his­ti­dine-tagged b9, untagged x, and untagged and N-ter­mi­nally hexa­his­ti­dine-tagged prin­ci­pal r sub­unit were con­ structed using rou­tine clon­ing meth­ods. The A. ae­o­li­cus rpo expres­ sion plas­mid set pro­vides a source of indi­vid­ual RNAP sub­units for in vitro recon­sti­tu­tion exper­i­ments.

Plas­mid pET28-AaeABZC, co-over­express­ing A. ae­o­li­cus rpoA, rpoB, rpoC, and rpoZ genes, was cre­ated accord­ing to a scheme pre­ sented in Fig. 2. First, two inter­me­di­ate plas­mids each con­tain­ing two genes—rpoA and rpoB (pET28-AaeAB), and rpoC + rpoZ (pET28AaeCZ)—were con­structed by (1) insert­ing the rpoA cas­sette from pET11-AaeA into pET28-AaeB and (2) insert­ing the rpoZ cas­sette from pET28-AaeZ into pET28-AaeC. The pET28-AaeABZC plas­mid was obtained by insert­ing the rpoA–rpoB cas­sette from pET28AaeAB into the pET28-AaeCZ plas­mid. pET28-AaeABZC con­tains four genes in the fol­low­ing sequence: rpoA, rpoB, rpoZ, and rpoC; each rpo gene is pre­ceded by T7 RNAP pro­moter. The rpoC gene is fused to the C-ter­mi­nal hexa­his­ti­dine tag and is fol­lowed by T7 tran­scrip­tion ter­mi­na­tor. Pre­vi­ous work in sev­eral sys­tems sug­ gests that C-ter­mi­nal tag­ging of b9 has no effect on RNAP activ­ ity and allows affin­ity puri­fi­ca­tion of RNAP and immo­bi­li­za­tion of func­tional tran­scrip­tion com­plexes. Table 2 lists A. ae­ol­i­cus rpo

Fig. 2. Con­struc­tion of a plas­mid co-express­ing A. ae­o­li­cus rpo genes. Steps involved in cre­a­tion of A. ae­o­li­cus rpo genes co-over­express­ing plas­mids are sche­mat­i­cally pre­ sented. See text for more details.



K. Ku­zn­ede­lov, K. Se­ver­i­nov / Methods 47 (2009) 44–52

Table 2 Aquifex ae­o­li­cus rpo genes and expres­sion plas­mids. Gene, plas­mid

Size (bp)

RNAP sub­unit

rpoA rpoB rpoC rpoD rpoZ pET11-AaeA pET28-AaeB pET28-AaeC pET11-AaeD pET28-AaeD pET28-AaeZ pET28-AaeAB pET28-AaeZC pET28-AaeABZC

954 4407 4725 1728 231 6272 9679 9964 7368 7060 5496 10784 10315 15954

a b b’ r x a b b9-His6-COOH r NH2-His6-r x a, b b9-His6-COOH, x a,b,b9-His6-COOH, x

genes PCR-ampli­fied from geno­mic DNA and plas­mids con­tain­ing these genes. 3.2. Prep­ar­ a­tion of A. ae­o­li­cus RNAP core enzyme To purify A. ae­ol­i­cus RNAP core from E. coli cells co-express­ing A. ae­o­li­cus rpo genes, BL21-Codon­Plus (DE3)-RIL (Strat­ag ­ ene) cells har­bor­ing pET28-AaeABZC were used to inoc­u­late 1 l of LB con­tain­ ing 25 lg/ml kana­my­cin and were grown at 37 °C for 18–20 h with vig­or­ous shak­ing. Cells (»4–5 g of wet bio­mass) were col­lected by cen­tri­fu­ga­tion (10 min at 4000g, 4 °C) and resus­pended in 40 ml of grind­ing buffer (Table 1) con­tain­ing 0.1 mM PMSF and 15 mM 2-ME. Cells were lysed by son­i­ca­tion and the debris was removed

49

by cen­tri­fu­ga­tion (30 min, 18,000g, 4 °C). The cleared cell lysate was trans­ferred into a 50 ml screw-cap poly­pro­pyl­ene cen­tri­fuge tube and incu­bated at 80 °C for 30 min with occa­sional mix­ing. Mas­sive pellet that was formed dur­ing this stage was removed by cen­tri­fu­ga­tion (30 min, 18,000g, 4 °C) and dis­carded. The super­na­ tant was loaded on a 5 ml Hep­a­rin Hi-Trap col­umn (GE Health­care) equil­i­brated in TGE buffer (Table 1) con­tain­ing 100 mM NaCl and 1 mM 2-ME. After load­ing, the col­umn was washed with TGE buffer con­tain­ing 300 mM NaCl, and RNAP was step-eluted in »7–10 ml of TGE buffer con­tain­ing 600 mM NaCl. Typ­i­cally ther­mo­phile results of SDS–PAGE anal­y­sis of frac­tions up to the 1 M NaCl hep­ a­rin col­umn elu­tion are shown in Fig. 3A. The 1 M NaCl hep­a­rin col­umn frac­tion was loaded on a 5 ml Hi-Trap che­lat­ing col­umn (GE Health­care) charged with Ni2+ and equil­i­brated in start buffer (Table 1) con­tain­ing 5 mM imid­az­ole. The col­umn was washed with the same buffer sup­ple­mented with 20 mM imid­az­ole, and A. ae­o­li­cus RNAP core was eluted with 100 mM imid­az­ole in the same buffer. Frac­tions con­tain­ing A. ae­o­li­cus RNAP core were pooled and pre­cip­i­tated over­night with pow­dered (NH4)2SO4 (0.3 g/ml) in a cold room. Ammo­nium sul­fate pellet was col­lected by cen­tri­fu­ga­ tion in a 15 ml poly­pro­pyl­ene cen­tri­fuge tube, drained thor­oughly, dis­solved in 250 lL of TGE buffer and loaded on a Su­pe­rose-6 HR 10/30 col­umn (GE Health­care) attached to an FPLC and equil­i­ brated in TGE con­tain­ing 200 mM NaCl and 1 mM 2-ME. A. ae­ol­i­cus RNAP eluted in two peaks. (Fig. 3B) The first peak con­tained mate­ rial strongly absorb­ing at 260 nm and prob­a­bly rep­re­sented RNAP com­plexes with nucleic acids and was dis­carded. The sec­ond peak con­tained pure (no less than 95% pure) RNAP core. This peak was col­lected, glyc­erol was added to the final con­cen­tra­tion of 50% and the enzyme was stored at ¡20 °C.

Fig. 3. Typ­i­cal puri­fi­ca­tion exam­ple of recombinant ther­mo­phile RNAP core enzyme from E. coli cells co-over­express­ing T. aquat­i­cus rpo genes. (A) SDS–PAGE anal­y­sis of pro­ teins in cell extract of induced co-over­express­ing cells (L), in a super­na­tant after heat-treat­ment (H), in a flow-through from a hep­a­rin-aga­rose col­umn (FT) and in frac­tions from the col­umn eluted with 0.3, 0.6, and 1.0 M NaCl in a buffer. A Coomassie-stained gel is shown. (B) Su­pe­rose-6 frac­tion­ation of T. aquat­i­cus RNAP core enzyme. Chro­mato­ graphic elu­tion pro­file, SDS–PAGE of indi­cated frac­tions are shown (lane labeled 0 con­tains mate­rial loaded on the col­umn). Mate­rial from the peak elut­ing early (E) and late (L) was pooled and ana­lyzed spec­tro­pho­to­met­ri­cally. The resul­tant spec­tra are shown on the right­hand side of the fig­ure.

50

K. Ku­zn­ede­lov, K. Se­ver­i­nov / Methods 47 (2009) 44–52

3.3. Aquifex ae­ol­i­cus r fac­tor prep­a­ra­tion

3.5. Tran­scrip­tion by ho­lo­en­zymes with inter­spe­cies r replace­ments

The pET28-AaeD plas­mid express­ing N-ter­mi­nally hexa­ his­ti­dine-tagged A. ae­o­li­cus r fac­tor was trans­formed in E. coli BL21(DE3) cells. Fresh trans­for­mants were inoc­ul­ ated in 1 l LB con­tain­ing 25 lg/ml kana­my­cin and grown at 37 °C. 0.5 mM IPTG was added when the cul­ture OD600 reached 0.7. After 6 h of growth with vig­or­ous agi­ta­tion at 37 °C, cells were har­vested and cell pellet (»1.5 g) was resus­pended in 10 ml of start buffer (Table 1) con­tain­ing 1 mM EDTA, 5 mM 2-ME, and 0.1 mM PMSF. Cells were lysed by son­i­ca­tion, cell debris removed by two cen­ tri­fu­ga­tion steps (30 min, 15,000g, 4 °C each) and the lysate vol­ ume was adjusted to 20 ml with the same buffer. The lysate was trans­ferred into a 50 ml screw-cap poly­pro­pyl­ene cen­tri­fuge tube and incu­bated for 30 min at 80 °C with occa­sional mix­ing. The mas­sive pellet was removed by low-speed cen­tri­fu­ga­tion (20 min, 15,000g, 4 °C) and dis­carded. Then super­na­tant was fil­ tered through Ac­ro­disc 25 mm Syringe Fil­ter with 0.45 lm Nylon Mem­brane (PALL, Life Sci­ences) and loaded onto a 1 ml Hi-Trap che­lat­ing col­umn (GE Health­care) charged with Ni2+ accord­ing to man­u­fac­turer’s instruc­tions and equil­i­brated in start buffer con­tain­ing 5 mM imid­az­ole. The col­umn was washed with the same buffer sup­ple­mented with 20 mM imid­az­ole, and A. ae­o­li­ cus sigma fac­tor was eluted with 100 mM imid­az­ole in the same buffer. Frac­tions con­tain­ing A. ae­o­li­cus r were pooled and con­ cen­trated on a cen­trif­u­gal fil­ter Am­icon Ultra (Mil­li­pore), dia­ lyzed against stor­age buffer (Table 1) and stored at ¡20 °C.

When hexa­his­ti­dine-tagged A. ae­o­li­cus r was puri­fied from E. coli extracts with­out the heat-treat­ment step, large amounts of E. coli RNAP core were always pres­ent in the 100 mM imid­az­ole frac­tion, sug­gest­ing that hybrid ho­lo­en­zymes form with high effi­ciency. No E. coli RNAP core is observed when recombinant T. aquat­i­cus r is puri­ fied. Pre­vi­ous work also sug­gested that while E. coli core was some­ what active with T. aquat­i­cus r, the reverse com­bi­na­tion was fully inac­tive [6]. We used abor­tive tran­scrip­tion ini­ti­at­ ion to check activ­ ity of hybrid ho­lo­en­zymes con­tain­ing RNAP core and r sub­units from A. ae­o­li­cus, E. coli, and T. aquat­i­cus. As can be seen from Fig. 4, the A. ae­o­li­cus RNAP core was only active with its cog­nate r fac­tor. In con­ trast, RNAP core from E. coli was active with all three r fac­tors tested, with activ­ity decreas­ing in the fol­low­ing order: E. coli r > A. ae­o­li­cus r > >T. aquat­i­cus r, sup­port­ing the idea that A. ae­o­li­cus r binds E. coli core bet­ter than T. aquat­i­cus r does. Of the two T. aquat­i­cus RNAP core-based hybrids, the one con­tain­ing E. coli r was inac­tive, while the one con­tain­ing A. ae­o­li­cus r exhib­ited good activ­ity lev­els.

3.4. In vitro tran­scrip­tion Tran­scrip­tion assays devel­oped for E. coli RNAP were used to test activ­ity of A. ae­o­li­cus RNAP with a sin­gle mod­i­fi­ca­tion: the reac­tion tem­per­a­ture was raised to 70 °C. Stan­dard abor­tive ini­ti­ a­tion reac­tions con­tained, in 10 ll of tran­scrip­tion buffer (Table 1), 50 nM A. ae­o­li­cus RNAP core enzyme and 100 nM of recombinant A. ae­o­li­cus r. Reac­tions were pre-incu­bated for 10 min at 70 °C, fol­lowed by the addi­tion of 100 nM T7 A1 pro­moter-con­tain­ing DNA frag­ment (¡54 + 92) and addi­tional 10- to 15-min incu­ba­ tion at 70 °C. Abor­tive tran­scrip­tion was ini­ti­ated by the addi­tion 100 lM CpA, 5 lM UTP, and 5 lCi a-[32P]UTP (3000 Ci/mmol), and allowed to pro­ceed for 10 min at 70 °C. Run-off tran­scrip­tion was ini­ti­ated by the addi­tion 100 lM CpA (or 0.5 mM ATP), 5 lCi a-[32P]UTP (3000 Ci/mmol) and 10 lM NTPs, and allowed to pro­ ceed for 10 min at 70 °C. Reac­tions were ter­mi­nated by the addi­ tion of equal vol­ume of form­am­ide-con­tain­ing load­ing buffer and ana­lyzed by dena­tur­ing gel-elec­tro­pho­re­sis (8 M urea, 20% poly­ acryl­amide) and auto­ra­di­og­ra­phy.

3.6. Anti­bi­otic sen­si­tiv­ity of A. ae­o­li­cus RNAP Despite the high degree of evo­lu­tion­ary con­ser­va­tion, bac­te­rial RNAPs exhibit dif­fer­en­tial sen­si­tiv­ity to anti­bi­ot­ics. This prop­erty must always be kept in mind when com­par­ing struc­tural data from avail­able Ther­mus RNAP struc­tures with genetic data obtained in other sys­tems, as erro­ne­ous inter­pre­ta­tions become pos­si­ble. We deter­mined the effect of two tran­scrip­tion inhib­i­tors, mi­cro­cin J (McJ) and rif­am­pi­cin (Rif) on in vitro activ­ity of A. ae­o­li­cus RNAP (McJ does not bind to and has no effect on Ther­mus RNAP; Ther­ mus RNAP behaves as a strong rif­am­pi­cin-resis­tant mutant of E. coli RNAP). With McJ, we hoped that if robust McJ inhi­bi­tion of A. ae­o­li­cus RNAP, a poten­tially crys­tal­liz­able enzyme, is observed, then struc­tural basis of tran­scrip­tion inhi­bi­tion could be inves­ti­ gated. This expec­ta­tion was not ful­filled, unfor­tu­nately (Fig. 5). At 0.1 mg/ml, McJ had little, if any, effect on A. ae­o­li­cus RNAP tran­ scrip­tion either in an abor­tive ini­ti­a­tion or in a steady-state runoff ­tran­scrip­tion assay. Tran­scrip­tion by the E. coli enzyme at these con­di­tions was strongly inhib­ited, as expected. The tran­scrip­tion tem­plate used in the exper­i­ment of Fig. 5 also con­tained an intrin­ sic tran­scrip­tion ter­mi­na­tor k tR2 between the tran­scrip­tion start site and the end of the tran­scrip­tion unit. As can be seen, the effi­ ciency of tran­scrip­tion ter­mi­na­tion (defined as the ratio of the amount of radio­ac­tiv­ity in the band of ter­mi­nated tran­script to the sum or radio­ac­tiv­ity in the ter­mi­nated and run-off tran­script bands) by the A. ae­o­li­cus RNAP was much lower than that of the E. coli enzyme. Sim­i­lar obser­va­tions were pre­vi­ously made for

Fig. 4. In vitro tran­scrip­tion by inter­spe­cies RNAP hybrids. The results of abor­tive ini­ti­a­tion from a T7 A1 pro­moter-con­tain­ing DNA frag­ment by ho­lo­en­zymes recon­sti­tuted from indi­cated core enzymes and r sub­units are pre­sented.



K. Ku­zn­ede­lov, K. Se­ver­i­nov / Methods 47 (2009) 44–52

51

Fig. 5. A. ae­o­li­cus RNAP is resis­tant to mi­cro­cin J. Results of abor­tive and run-off tran­scrip­tion from a T7 A1-pro­moter tem­plate in the pres­ence or in the absence of McJ are pre­sented.

Fig. 6. Tran­scrip­tion inhi­bi­tion by rif­am­pi­cin. The results of steady-state tran­scrip­tion by the indi­cated enzymes in the pres­ence of indi­cated con­cen­tra­tions of rif­am­pi­cin are shown.

52

K. Ku­zn­ede­lov, K. Se­ver­i­nov / Methods 47 (2009) 44–52

­ her­mus RNAP, sug­gest­ing that ther­mo­philic RNAPs may be less T effec­tive in rec­og­ni­tion of tran­scrip­tion ter­mi­na­tors. Another com­pound tested was rif­am­pi­cin, a clas­si­cal inhib­i­tor of bac­te­rial RNAP. While most stud­ies sug­gest that Rif is a sim­ ple ste­ric inhib­i­tor that blocks the exten­sion of nascent tran­scripts beyond the length of 2–3 nucle­o­tides [13], struc­tural work with T. ther­mo­phi­lus RNAP led to more com­plex hypo­thet­ic­ al mech­a­nisms, involv­ing al­los­ter­i­cal effects of Rif on the RNAP cat­al­ ytic cen­ter [14] (the con­clu­sions of the lat­ter paper were very recently seri­ously chal­lenged and may not be valid, [15]). In the case of E. coli RNAP, Rif behaved in an expected way, inhib­it­ing, at low con­cen­tra­tions, the pro­duc­tion of run-off tran­scripts while strongly stim­u­lat­ing the pro­duc­tion of abor­tive tri­mer CpAp­U (Fig. 6, note that in the gel sys­tem used here, the tri­mer has a lower mobil­ity than the CpA­pUpC tet­ra­mer). In the case of T. aquat­ic­ us enzyme, increas­ing con­cen­tra­tions of Rif lead to pro­gres­sively lower lev­els of run-off tran­scripts pro­duc­tion (which how­ever was never fully inhib­ited), with little or no effect on the CpAp­U pro­duc­tion. The case of A. ae­o­li­cus RNAP pre­sented yet another pattern of inhi­bi­tion. Rif had little effect on run-off tran­scripts pro­duc­tion, while clearly stim­u­ lat­ing abor­tive tran­scrip­tion. Such pattern may be con­sis­tent with revers­ible bind­ing of Rif to the enzyme (high off rate). In con­trast, in the case of T. aquat­i­cus enzyme, the on rate of Rif bind­ing may be low, but the bind­ing itself is sta­ble. Be that as it may, the observed dif­fer­ences sug­gest that the detailed mech­a­nism of Rif action may be dif­fer­ent in dif­fer­ent sys­tems, call­ing for cau­tion when com­par­ ing func­tional and struc­tural data obtained with RNAPs from dif­ fer­ent ori­gins. 4. Con­clu­sions The pro­to­cols pre­sented here were suc­cess­fully used for RNAP prep­a­ra­tion from var­i­ous eu­bac­te­ria. We believe that the same pro­to­cols can be used with minor mod­ifi ­ ­ca­tion for prep­a­ra­tion of RNAP from any bac­te­ria. The avail­abil­ity of vir­tu­ally unlim­ited amounts of bac­te­rial RNAPs, cou­pled with mod­ern pro­teo­mic tools

avail­able for iden­ti­fic­ a­tion of RNAP-bind­ing pro­teins and bio­in­for­ mat­ics tools for pre­dic­tion of puta­tive pro­mot­ers shall allow iden­ ti­fi­ca­tion of novel tran­scrip­tion reg­u­la­tory mech­a­nisms that are dis­tinct from those oper­a­tional in E. coli in the near future. Acknowl­edg­ments The pro­ject described above was sup­ported by NIH RO1 Grants GM59295 and GM64503 and a Rus­sian Acad­emy of Sci­ences Pre­ sid­ium pro­gram grant in Cell and Molec­u­lar Biol­ogy to K.S. Ref­er­ences [1] S. Bor­uk­hov, A. Gold­farb, Prot. Exp. Purif. 4 (1993) 503–511. [2] H. Tang, K. Se­ver­i­nov, A. Gold­farb, R.H. Ebright, Proc. Natl. Acad. Sci. USA 92 (1995) 4902–4906. [3] L. Min­ak­hin, S. Ne­chaev, E.A. Camp­bell, K. Se­ver­i­nov, J. Bac­te­riol. 183 (2001) 71–76. [4] G. Zhang, E. Camp­bell, L. Min­ak­hin, C. Rich­ter, K. Se­ver­i­nov, S.A. Darst, Cell 98 (1999) 811–824. [5] K. Ku­zn­ede­lov, N. Kor­zh­eva, A. Mus­taev, K. Se­ver­i­nov, EMBO J. 21 (2002) 1369– 1378. [6] K. Ku­zn­ede­lov, L. Min­ak­hin, K. Se­ver­i­nov, Meth­ods Enz­y­mol. 370 (2003) 94– 108. [7] K. Ku­zn­ede­lov, V. Lamour, G. Pat­i­kog­lou, M. Chle­nov, S.A. Darst, K. Se­ver­i­nov, J. Mol. Biol. 359 (2006) 110–121. [8] T. Nary­shk­in­a, K. Ku­zn­ede­lov, K. Se­ver­i­nov, J. Mol. Biol. 361 (2006) 634–643. [9] I. Ar­tsi­mov­itch, V. Svet­lov, K.S. Mura­ka­mi, R. Lan­dick, J. Biol. Chem. 278 (2003) 12344–12355. [10] E.C. Ne­dea, D. Mar­kov, T. Nary­shk­in­a, K. Se­ver­i­nov, J. Bac­te­riol. 181 (1999) 2663–2665. [11] K. I­gar­ash­i, N. Fuj­it­a, A. Ishi­ha­ma, Nucleic Acids Res. 18 (1990) 5945–5948. [12] G. Deck­ert, P.V. War­ren, T. Ga­as­ter­land, W.G. Young, A.L. Lenox, D.E. Gra­ham, R. Over­beek, M.A. Snead, M. Kel­ler, M. Au­jay, R. Hu­ber, R.A. Feld­man, J.M. Short, G.J. Ol­sen, R.V. Swan­son, Nature 392 (1998) 353–358. [13] E. Camp­bell, N. Kor­zh­eva, A. Mus­taev, K. Mura­ka­mi, S. Nair, A. Gold­farb, S.A. Darst, Cell 104 (2001) 901–912. [14] I. Ar­tsi­mov­itch, M.N. Vas­syly­eva, D. Svet­lov, V. Svet­lov, A. Perede­ri­na, N. I­gar­ ash­i, N. Mats­ugaki, S. Wa­kat­su­ki, T.H. Ta­hi­rov, D.G. Vass­ylyev, Cell 122 (2005) 351–363. [15] A. Fek­lis­tov, V. Me­kler, Q. Ji­ang, L.F. West­blade, H. Irsc­hik, R. Jan­sen, A. Mus­ taev, S.A. Darst, R.H. Ebright, Proc. Natl. Acad. Sci. USA 105 (2008) 14820– 14825.