The Yeast Saccharomyces Cerevisiae
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Proc. Nati. Acad. Sci. USA Vol. 86, pp. 8348-8352, November 1989 Biochemistry Transcription terminates near the poly(A) site in the CYCI gene of the yeast Saccharomyces cerevisiae (mRNA termination/mRNA precursor/polyadenylylation/iso-l-cytochrome c) PATRICK Russo*t AND FRED SHERMAN*t§ Departments of *Biochemistry and tBiophysics, University of Rochester Medical School, Rochester, NY 14642 Contributed by Fred Sherman, August 7, 1989 ABSTRACT A 38-base-pair region required for normal MATERIALS AND METHODS CYC) mRNA 3' end formation in Sacclharomyces cerevisiae was Oligonucleotides. Oligonucleotides used for site-directed shown to be necessary for the termination of transcription in mutagenesis and probing Northern blots were synthesized on vivo by examining the stability of CEN3 plasmids. CEN3 an Applied Biosystems 380A DNA synthesizer. plasmids were stably maintained during vegetative growth, Plasmid Construction. The 1.3-kbp EcoRI-BamHI restric- unless a GAL) transcript impinged on the CEN3 region. tion fragment of the plasmid pGALCEN3 (9), containing the Transcription from the GAL] promoter was terminated, and GALJO and GALI promoters and CEN3 element (Fig. 1A), plasmid stability was restored by the insertion of a fragment was transferred to plasmid pEMBL8 (10). All of the con- containing the 38-base-pair region of CYCI. In contrast, a structs listed below were made in the above-mentioned similar fragment lacking the 38-base-pair region had no such derivative of pEMBL8 and then transferred back into stabilizing effect. Furthermore, CYCI mRNA transcription pGALCEN3 as an EcoRI-BamHI fragment, generating the terminated in a region <100 nucleotides downstream from the plasmid series pAB610 through pAB615 (Fig. 1 B and C). normal poly(A) site, thus establishing that CYCI mRNA 3' end A Xho I site was created between the GALl promoter and formation does not involve overly extended precursors as are CEN3 element by using the oligonucleotide site-directed observed in higher eukaryotes. mutagenesis method of Kunkel et al. (11). The Xho I site was positioned 33 nt upstream from the CDE I (centromere DNA The proper maturation ofmRNA 3' ends in higher eukaryotes element I) component of CEN3 and 83 nt downstream from is an important step in gene expression. Single point muta- the GAL] transcription initiation site. The pGALCEN3 de- tions in the higher eukaryotic mRNA 3' end processing rivative containing the newly created Xho I site was desig- signal, AAUAAA, can lead to a drastic decrease in gene nated pAB610 (Fig. 1B). expression caused by inefficient cleavage and polyadenylyl- Different DNA fragments from the 3' regions ofthe CYCI+ ation of precursor transcripts (1, 2). There is also evidence and cycl-512 genes were inserted in the Xho I site between that the AAUAAA cleavage signal assists in the downstream the GAL] promoter and CEN3 element. Plasmid pAB611 termination ofthe precursor mRNAs in higher eukaryotes (3, contains the 468-bp EcoRV-Sal I fragment from CYCI + (Fig. 4). This relationship between transcript cleavage and down- 2A) in the orientation in which it is transcribed in the stream transcription termination also may be operating in the wild-type CYCI locus. Plasmid pAB615 contains the same yeast Saccharomyces cerevisiae. EcoRV-Sal I fragment of pAB611, but in the opposite Unlike higher eukaryotes, yeast do not appear to use the orientation. Plasmid pAB612 contains the 430-bp EcoRV-Sal AAUAAA signal but use other DNA-encoded signals for 3' I fragment from the 3' region of the cycl-512 locus. Plasmid end formation (5-7). Butler and Platt (8) demonstrated that in pAB612 is similar to pAB611, except that pAB612 lacks the vitro generated CYCI and UTRI mRNAs, which extended 38-bp sequence represented in Fig. 2B. Plasmid pAB613 was past their normal 3' end points, were cleaved and polyaden- generated from pAB611 by removing the 219-bp HindIII-Sal ylylated at or near their wild-type poly(A) sites with a yeast I fragment, leaving a 249-bp EcoRV-HindIII fragment be- cell-free lysate. In the case of CYCI, the in vitro 3' end tween GALI and CEN3. Plasmid pAB614 contains the 83-bp processing was dependent on a 38-base-pair (bp) region, 8 Ava II-FnuDII fragment from the 3' region of CYCI (Fig. nucleotides (nt) upstream from the poly(A) site. This same 2A). This Ava II-FnuDII fragment contains a Xho I linker at 38-bp region was originally identified by Zaret and Sherman the Ava II end and a Sal I site at the FnuDII end and was (5) as the cycl-512 deletion that caused aberrantly long CYCI kindly supplied by Steven Baker (University of Rochester). transcripts. If the sole function of sequences within the 38-bp Plasmid Stability Assay. The yeast strain J17 (9) was region were to define a cleavage point, then the extended transformed with plasmids pAB610 through pAB615 by the of would precursor lithium acetate method of Ito et al. (12). These yeast strains transcripts cycl-512 represent CYCJ containing one of the plasmids (Fig. 1) were pre-grown in mRNAs. This paper presents evidence that the extended minus-uracil medium [consisting of 0.67% yeast nitrogen cycl-512 transcripts are not the true precursors in vivo. In base (without amino acids); 2% dextrose; adenine sulfate, addition, we demonstrate that CYCI transcription terminates L-arginine hydrochloride, L-histidine hydrochloride, L- within 100 nt of the mRNA poly(A) site in vivo and that this methionine, and L-tryptophan, each at 20 mg/liter; L- termination is dependent on the same 38-bp region involved isoleucine, L-leucine, L-lysine hydrochloride, and L-tyrosine, in 3' end processing in vitro. Thus mRNA 3' end processing each at 30 mg/liter; L-phenylalanine at 50 mg/liter; and and transcription termination are interrelated in yeast, as in L-valine at 150 mg/liter]. Cells from the minus-uracil pre- higher eukaryotes, but with a different set of DNA-encoded cultures were used to inoculate glucose liquid medium (con- recognition signals and spatial dimensions. Abbreviation: nt, nucleotide(s). The publication costs of this article were defrayed in part by page charge tPresent address: Institute of Biochemistry, University of Helsinki, payment. This article must therefore be hereby marked "advertisement" Valimotie 7 SF-00380, Helsinki, Finland. in accordance with 18 U.S.C. §1734 solely to indicate this fact. §To whom reprint requests should be addressed. 8348 Downloaded by guest on September 28, 2021 Biochemistry: Russo and Sherman Proc. Natl. Acad. Sci. USA 86 (1989) 8349 A URA3 ARSI 4- -+ CDE pGALCEN3 iAMpR GALIO GALI ium ECoU , I. BamiFU ... % Loss B generation GALI Transcription initiation XhoI CDEI pAB610 33 bp - 8% Insertions j C F -( 468bp 1<318bp 1 nrlSail <~I 219 bp I~ - II pAB61l1 ~ -- I 3.5%/6 EcoRV PolywA Htnd IlI Sall 38 bp Deletion pAB612 I- PI 9% EcoRV Polw HudnII Sall I 249bp- - Term. Signal PAB613 I 4.5% EcoRV Poly" HtndIfl <- 83 bp -- Term. Signal pAB614 12% ALaII PolyA FhuDII Term. Signal pAB615 L- I~E IPCo 100/0 SaHl Hin~dMI PolytA EcRV FIG. 1. Structures and stabilities of plasmids. (A) Plasmid pGALCEN3 (9), showing the Escherichia coli ampicillin-resistance (AmpR) marker and the yeast URA3 gene, ARSJ replicating region, GALJO and GALI promoters, and CEN3 centromeric region, containing CDE 1, CDE II, and CDE III. (B) Plasmid pAB610, showing the Xho I site that was created in the pGALCEN3 plasmid between the GAL] promoter and CEN3 element. (C) Plasmids pAB611 through pAB615, containing various DNA segments from the CYCJ + and cycl-512 3' mRNA regions inserted in the Xho I site of pAB610. Plasmid pAB611 contains the 468-bp EcoRV-Sal I fragment from CYCJ in an analogous wild-type orientation, shown in Fig. 2A. Plasmid pAB612 contains the corresponding fragments from the cycl-512 mutant, which lacks the mRNA 3' end-forming signal because of a 38-bp deletion. Plasmid pAB613 was generated from pAB611 by removing the 219-bp HindIII-Sal I fragment. Plasmid pAB614 contains the 83-bp Ava II-FnuDII fragment flanked on the Ava II end by a Xho I linker and on the FnuDII side by a Sal I site. Plasmid pAB615 contains the same EcoRV-Sal I fragment inserted in pAB611 but in the opposite orientation. The percent loss per generation presented at right was calculated from the results presented in Fig. 3. sisting of 1% Difco yeast extract, 2% Difco peptone, and 2% otide kinase reaction of Maxam and Gilbert (14). Hybridiza- dextrose) and galactose liquid medium (consisting of 1% tion to the probes was performed at room temperature for Difco yeast extract, 2% Difco peptone, and 2% galactose). 8-12 hr. The blots were washed with 0.30 M sodium chloride/ Portions from the growing cultures were removed at various 0.03 M sodium citrate/0.1% SDS and then exposed to x-ray times, diluted, and plated on both glucose medium, for film with the aid of an intensifying screen. Probes were determining total cell number (Ura' and Ura-), and minus- washed off the membranes at 650C in the prehybridization uracil medium, for determining Ura' cell number. buffer mentioned above for 1 hr, prior to hybridization to Northern Analysis. Total RNA was extracted (5) from yeast other oligonucleotide probes. J17 containing plasmids pAB610 through pAB615 that had been grown in galactose medium. Twenty-five micrograms of RESULTS RNA was loaded per lane of a 1.5% agarose gel containing 3% formaldehyde and lx Mops buffer (20 mM 4-morpholino- Termination of Transcription by a 3' Fragment from the propanesulfonate, pH 7.0/5 mM sodium acetate/1 mM CYCI Gene. Hill and Bloom (9) showed that the mitotic EDTA).