New RNA Polymerase from Escherichia coli infected with Bacteriophage T7

A coupled in vitro transcription‐translation system for the exclusive synthesis of polypeptides expressed from the T7 promoter

“…track g), but IS rapldly degraded m the oidinary S30 (trucks a-f). In contrast, pYCP2 is Incubdtlon wds cdrned out dt 37°C m the dbsencc OF rlfdmplcm Sdmplc5 were removed dt venous tlmc mtcrvdls (0 15, 30,45, 60, 100 n1i11, trdcks 3-8 drid trdcks d-f, rcspcctWiy) dnd dddcd to dn equal volume of dgnlosc gel snmplc buffer (contdmmg 4% w/v SDS) The sdmplcs were dlldly5rd by dgdrcse gel elcctlophoresls on d 1% (w/v) gel then sldmcd with cthldntm bromldc (0 5 m&ml) tor 30 mm For rcfcrcncc trdck I shows cxtrdct wlthout ddded DNA nntl trdck 2 shows 0 5 irg 01 the rclcvdnt pldsmld tcmpldtc Trdcb H shows ~H~,ldlII stdnddrds rapldly degraded m both extracts (Fig, 6, bottom photograph) Therefore, lmc,lr DNA only surv~es well when <I T7 promoter 15 present on the plasmld and T7 RNA polymcrasc IS present m the cxtr,tct T7 RNA polymcrase elongdtcs RNA chains five tunes faster than E colr RNA polymcruse [2] and so it IS possible that the incleascd frcqucncy of tl,mscnptlon prcvcnts dcgradatlon of the IlIIcal tcmplatc Therefore, m the presence of T7 KNA polymcrase, a DNA tcmplstc encodmg the T7 promoter (and thcrcforc rccogmscd by T7 RNA poly mclosc) 15 more &lblc th,ui DNA not lccogniscd by T7 RNA p~lymer~~~. This cxplillns why less lmcar DNA FEBS Em-TER.S Octolxr 1991 was required to productively &me the T7-SSO extract…”

“…T7 TNA polymerase exhibits extreme specificity for its own promoter and will efficiently transcribe any DNA cloned downstream of the T7 promoter [I], An S30 extract containing endogenous T7 RNA polymerase (T7630) was preparLd and optimised for use in vitro. Rifampicin was added to the system in order to selectively inhibit & co/i RNA polymcrase [2] and under such conditions. when primed with a plasmid template (encoding the target gene under exclusive control of the T7 promoter), the system can be used to synthesise unique, labelled target protein, In this paper we dcmonstrate the preparation, use and characterisation of this in vitro system.…”

“…SDS-PAGE analysis showed that as little as 25 ng ( Fig 2, track 3) of supercoIled pMJ2 I6 was suffiaent to dlrect polypeptlde synthesis, compared with 250 ng m the normal extract (unpubhshed results) Slmdarly with a lmear template, ordmary extract rcqnred In excess of I ,I.J~ of hncar DNA (unpublished results), whereas with the T7-S30 extract, 500 ng (Fig 2, track 13) of hncar DNA was sufficlcnt Moreover, T7-S30 extract exhlblted httle. if any, background mcorporatlon, demonstratmg that no endogcnous r&WA and DNA was present m the extract Normally extracts dre prepared from plasmld-free strams and so it was possible that plasmld pARI could have survived the extract procedule to act as an endogenous template However, In T7 RNA polymerasc-spcclfic extracts, endogenous DNA templates would not be such a problem since host det lved template expresslon cdn be sclectlvely inhibited by rlfdmplcm [2] In order to determme the concentration of rlfamplcm needed to mhlblt transcrlptlon by E COIL RNA polymerase m the extract, plasmld pLB8000 (-T7 ptomoter) was used as a template The addltlon of methanol alone (0 7% v/v final concentlatlon, Fig 3, track 2) had no effect upon expression, but mcrcasmg concentrations of rlfamplcm (m methanol, Fig 3. colr RNA polymerase and therefore abohshcd protem synthesis I-Iowever, on longer exposure of the autoradlograph fault bands were vlslble rn tracks 3, 4, and 5 of Fig 3 (3, 13 and 30 pug/ml final concentration of nfamplcm, respcctlvcly) and so for total mhlbltlon, a final concentration of 350 pglml of Ilfampicm (2 pl of 10 mg/ml rlfamplcm m 10% methanol, Fig 3, track 7) was added to each subsequent mcubatlon…”

Enzymes used in molecular biology: a useful guide

“…SP6 RNA polymerase is a 96 kDa polypeptide purified from SP6 bacteriophage-infected Salmonella typhimurium LT2 (Butler and Chamberlin 1982), while T7 RNA polymerase is a 98 kDa polypeptide (Stahl and Zinn 1981) produced by the T7 bacteriophage (Chamberlin et al 1970). RNA polymerases are used for in vitro synthesis of anti-sense RNA transcripts , production of labeled RNA probes, or for RNase protection mapping (Zinn et al 1983).…”

Coliphage BA14: a New Relative of Phage T7

“…The supernatant fluid was used as enzyme source. The following procedures were then employed, with minor modifications: the method of Gefter et al (1966) for assaying S-adenosyl-methioninecleaving enzyme (SAMase) activity; the method of Chamberlin et al (1970) for assaying RNA polymerase activity; the method of Rahmsdorf et al (1974) for assaying protein kinase activity.…”