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Translation Activates the Paused Transcription Complex and Restores Proc. Nadl. Acad. Sci. USA Vol. 82, pp. 4663-4667, July 1985 Biochemistry Translation activates the paused transcription complex and restores transcription of the trp operon leader region (tanscripfton-tnlation coupling/transcrIptIon pausing/attenuation/pause complex half-life) ROBERT LANDICK, JANNETrE CAREY*, AND CHARLES YANOFSKY Department of Biological Sciences, Stanford University, Stanford, CA 94305 Contributed by Charles Yanofsky, April 5, 1985 ABSTRACT It has been proposed that RNA polymerase have been identified in the trp (6-11), ilv (12), and thr (13) pausing in the leader region of the tryptophan (tIp) operon of operon leader regions, at the position corresponding to the 3' Escherichia coli is responsible for the synchronization of end of the first RNA hairpin. If the paused RNA polymerase transcription and translation essential to attenuation control. remained at this site until ribosome movement caused re- In this report we use an in vitro coupled transcription/ sumption of transcription, synchronization of transcription translation system to study the effect of tip leader peptide and translation would be accomplished. synthesis on RNA polymerase pausing in the tip leader region. Previous studies with the trp operon demonstrated that (l) Wild-type and translation-defective tip leader templates of E. the predicted stability of the pause RNA hairpin is directly cofi and Sernitia marcescens were employed, and pause RNA correlated with the strength of the pause signal (10, 11); (it) synthesis and paused complex release (activation) were quan- NusA protein enhances pausing by RNA polymerase (9, 10, tified relative to synthesis of the terminated leader transcript. 14); (iih) low concentrations of GTP but not the other NTPs It was observed that pausing in the tip leader region was (guanylic acid is the next nucleotide to be added) also prolonged when translation of the leader transcript was re- enhance pausing (6, 10, 11); (iv) mutant RNA polymerases duced by mutations in the leader region or by addition of the that increase or decrease transcription termination at the trp translation inhibitor kasugamycin or chloramphenicol. Exper- attenuator have analogous effects on pausing (4, 8); and (v) iments with S-30 extracts from a mutant strain that is ineffi- RNA polymerase pausing at the trp leader pause site can be cient in translating the tryptophan codons in the leader detected in a coupled transcription/translation system (11). transcript indicated that ribosome movement to these codons In this report we use the coupled system to show that the also releases the paused transcription complex. These findings ribosome engaged in synthesizing the leader peptide releases indicate that the paused tip leader transcription complex the paused complex.! resumes transcription when released by ribosome movement over the leader peptide coding region. This release would MATERIALS AND METHODS facilitate the coupling of transcription and translation essential to attenuation control. Nucleoside triphosphates, rifampicin, kasugamycin, chlor- amphenicol, and reagents required for cell-free transcription/ Transcription attenuation regulates expression of many ami- translation reactions were purchased from Sigma. [a- no acid biosynthetic operons of enteric bacteria (for recent 32PJGTP and [35S]methionine were from Amersham. RNA reviews, see refs. 1-4). According to the current model of polymerase and tryptophanyl-tRNA synthetase were gener- attenuation, formation ofalternative secondary structures in ous gifts of R. Fisher and K. Muench, respectively. Plasmid the transcript of the leader region controls transcription DNAs used as templates for S-30 reactions were prepared by termination at a site immediately preceding the structural standard procedures (15, 16). Restriction fragments used for genes of the operon. It is believed that translation of a short in vitro transcription reactions were prepared by Sau3A peptide coding region in the leader transcript governs for- (Escherichia coli templates) or Hpa II (Serratia marcescens mation of these alternative transcript secondary structures. templates) digestion of appropriate plasmids (17, 18, 19) and In the trp operons ofall enteric bacteria examined the leader isolated from polyacrylamide gels. S-30 cell-free extracts peptide coding region contains tandem tryptophan codons were prepared from RNaseI- strains as described (20, 21, 22). (5). Ribosome stalling at either ofthese tryptophan codons is In Vitro Transcription (Noncoupled). Paused complex half- believed to promote formation of a transcript secondary lives were determined in synchronized single-round in vitro structure, the antiterminator, that prevents transcription transcription experiments conducted as described (6, 9, 10). termination in the leader region. However, when a ribosome Paused complex half-lives were calculated by linear regres- translates the entire leader peptide coding region without sion analysis of the logarithm of pause RNA concentrations stalling it promotes formation of a different transcript sec- vs. time. ondary structure, the terminator. This secondary structure Coupled Transcription/Translation Reactions. Cell-free causes transcription termination at the attenuator in the transcription/translation experiments were performed essen- leader region. The attenuation model demands that transla- tially as described (20). To measure RNA synthesis unlabeled tion of the leader peptide coding region of the transcript be GTP was replaced by [a-32P]GTP. To measure protein closely coupled to transcription of the leader region. This synthesis unlabeled methionine was replaced by [35S]methi- coupling could be achieved if the polymerase molecule onine. Plasmid DNAs were added to a final concentration of transcribing the leader region paused until it was approached 40 nM. The translation inhibitors chloramphenicol and by the ribosome engaged in translating the leader peptide coding region of the transcript. Transcription pause sites *Present address: Department ofBiochemistry, Stanford University Medical School, Stanford, CA 94305. tIn this paper we use "release of the paused complex" or "paused The publication costs ofthis article were defrayed in part by page charge complex release" to mean that RNA polymerase has resumed payment. This article must therefore be hereby marked "advertisement" transcription elongation after having paused at the trp leader pause in accordance with 18 U.S.C. §1734 solely to indicate this fact. site. 4663 Downloaded by guest on October 2, 2021 4664 Biochemistry: Landick et aL Proc. Natl. Acad Sci. USA 82 (1985) kasugamycin were added to the cell-free extract prior to 1:2 A A mixing with the other reaction components. This order of A A STOP G=C mixing was necessary to achieve even 50%o inhibition of U -G C=G protein synthesis by kasugamycin. Reactions were initiated AAGUUCACG SER C-G by adding the cell-free extract (20% of final volume) and A U-A A THR C=G shifting the mixture to 370C. After 15 min of incubation to A A-U A C=G allow transcription and translation to reach steady-state A ARG G U G c A activity, [a-32P]GTP was added. In time course experiments, U G G U after a 2-min labeling period rifampicin was added to block U TRP G=C A U-A further transcription initiation. Samples were removed and U TRP G=C added to 0.6 vol of phenol at the times indicated. To measure C METLYSALAILEPHEVALLEULYSGLYG=C GACAAUAAAGCAAUUUUCGUACUGAAAGGUU-AU * () steady-state pause RNA levels, the reactions were stopped A with 0.6 vol of phenol after the 2-min labeling period. After T RPL29 centrifugation, the aqueous layers of samples were added to an equal volume of transcription stop mix (23) and electro- B phoresed on 10% polyacrylamide gels containing 7 M urea. A310--lA'.'I A C The relative pause RNA level was determined by counting A G the Cerenkov radiation of gel slices and calculating the molar C=G percent of pause RNA in the total of pause plus leader RNAs C=f=c- -A221- = A-b 1:2 (relative pause RNA level molar percent of pause RNA in AAGUUC=GUCGGAU LEU the total ofpause plus leader RNAs). The relative pause RNA CUG LEU U C level is an arbitrary steady-state measure of the half-life for C=G ARG C=G release of paused transcription complexes and is dependent SER C=G on the length of the labeling period (2 min for all measure- U C-UC ALA IHR C-U ments). Because of the complex kinetics of transcription in A-U VAL the coupled system, we used the relative pause RNA level AUG calculation to simplify analysis of coupled transcription/ ARG A STOP measure A translation pausing data. One complication of this is TRP GUC that transcription read-through at the attenuator influences METASNTHRTYRILESERLEUHISGLYTRP C-G the amount of leader RNA. By using a template containing a GUCUGCAAAUGAACACAUACAUUUCUCUUCACGGUUGGUGG=C (G) strong transcription terminator downstream from the trp FIG. 1. Sequence and secondary structure of trp leader pause attenuator to yield a read-through product of defined length, RNAs. (A) RNA secondary structure predicted for the E. coli trp we estimated the level of read-through at the E. coli trp pause RNA. RNA polymerase pausing occurs at the position marked attenuator to be -4% under our assay conditions. Except for by the boldface arrow. The G -+ A change that is present in trpL29 certain situations reported below, this level did not vary is indicated. Structure 1:2 is thought to be the primary pause signal between wild-type and trpL29 templates or with (10). The G shown in parentheses is the next
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