A novel suppressor tRNA from the dimorphic fungus Candida albicans

Biochirnica et Biophvsica Acta 866 (1986) 26-31 Elsevier

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BBA91555

A novel s u p p r e s s o r t R N A f r o m the dimorphic f u n g u s Candida albicans M i c h a e l F. T u i t e a, P a t r i c i a A. B o w e r b a n d C a l v i n S. M c L a u g h l i n b a Biological Laboratory, University of Kent, Canterburp, Kent, CT2 7NJ (U.K.) and b Department of Biological Chemistry, University of California, Irvine, lrvine, CA 92717 (U.S.A.) (Received September 9th, 1985)

Key words: Mistranslation; Suppressor tRNA; (C. albicans)

Unfractionated tRNAs from a number of prokaryotes and eukaryotes were examined for their ability to promote termination codon readthrough in a cell-free system isolated from Saccharomyces cerevisiae, tRNA from the dimorphic fungus Candida albicans was found to have significant UGA and UAG readthrough activity and this activity was present in tRNA extracted from both the yeast and the hyphai phase of the fungus. Unusually the efficiency of readthrough activity in vitro was not affected by the [psi] determinant. C. albicans tRNA was fractionated by one-dimensional and two-dimensional gel electrophoresis and both readthrough activities appeared to be associated with a single species of tRNA. Introduction

Materials and Methods

Increasing evidence from both eukaryotic and prokaryotic organisms has suggested that the regulated translational readthrough of termination codons, in particular the UGA codon, may define a novel mechanism for regulating gene expression [1,2]. Transfer RNAs (tRNAs), present in wild-type cells, that are able to translate the UGA codon have been identified in Escherichia coli [3], Saccharomyces cerevisiae [4,5], rabbit reticulocytes [6], wheat germ [7] and bovine liver [8]. Similarly, wild-type tRNAs able to translate the UAG (amber) codon in vitro have been identified in wheat germ [9], Drosophila melanogaster [10] and tobacco plants [11]. We have examined the ability of unfractionated tRNAs, isolated from a number of different organisms, to translate either the U G A codon or the U A G codon, in a cell-free system prepared from S. cerevisiae. We report here the identification of a single tRNA from the dimorphic fungus Candida albicans that is able to translate efficiently both the UAG and the U G A codon, as well as inducing a more general in vitro mistranslation of natural mRNAs.

Cell-free translation. Cell-free lysates were prepared from a haploid strain of S. cerevisiae (465/4d: M A T a ade2-1 lysl-1 can 1-100 ura3-1 [psi +]). The method of preparation and the conditions used to translate natural mRNAs have been previously described [12]. Cell-free translation products were labelled with [35S]methionine ( > 1000 C i / m m o l ) and analysed on 17.5% SDS-polyacrylamide gels as previously described [12]. tRNA isolation, tRNA was isolated from stationary-phase yeast cells of C. albicans as previously described [13], and separation by two-dimensional polyacrylamide gel electrophoresis was essentially as described by Fradin et al. [14]. Gels were stained with aqueous methylene blue for a short period to visualize the separated tRNAs, which were then eluted as follows; a piece of gel containing the tRNA(s) of interest was cut out, crushed with a sterile glass rod, and resuspended in 0.5% SDS, 0.3 M NaC1, 0.01 M Tris-HC1 (pH 7.4). Following incubation at 37°C for 1 h, the gel slurry was centrifuged through siliconized glass wool to remove solid material and the tRNA was

0167-4781/86/$03.50 © 1986 Elsevier Science Publishers B.V. (Biomedical Division)

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precipitated with 2.5 vol. of 95% ethanol at - 20°C. The precipitated tRNA was taken up in 5 #1 of water prior to addition to the translation system. Results

Total tRNAs, isolated from a number of different organisms, were examined for their ability to allow in vitro translation to continue past natural UGA and UAG termination codons. Such termination readthrough can be assayed in vitro by examining readthrough of the rabbit fl-globin UGA terminator [13,15,16] and the Brome Mosaic Virus (BMV) coat protein UAG terminator [13]. In each case an elongated polypeptide is synthesized which can be separated electrophoretically from the normally terminated translation product, and thus allows a quantitation of the efficiency of readthrough. We have previously shown [4,13] that

a.

such a system can be used to assay yeast nonsense suppressors in a homologous cell-free system. As shown in Table I, a number of tRNA samples examined contained UGA suppressor activity, with the exception of tRNA from wheat and soybean. The UGA suppressor activities in E. coli [3], S. cerevisiae [4] and rabbit reticulocyte [6] have been previously described. In S. cerevisiae the major endogenous UGA suppressor activity comes from a mitochondrial tRNATw [4,5]. tRNAs from both strains of C. albicans examined (304 and C9) had significant UGA suppressor activity and were also capable of inducing UAG readthrough (Table I). Of the other tRNAs examined only tRNA from an S. cerevisiae strain carrying the SUP61 amber suppressor mutation gave detectable UAG readthrough. Fig. 1 shows the in vitro products synthesized in the presence and in the absence of the C.

b.

H

13

*--CC T

Fig. 1. Readthrough of U A G and U G A natural termination codons induced by C albicans t R N A in a yeast cell-free system. (a) Rabbit globin m R N A (400 p,g/ml) was translated in vitro and tRNAs, added at a final concentration of 80 p,g/ml, were as follows; lane 1, no tRNA; lane 2, tRNA from a sup + [rho + ] strain of S. cerevisiae; lane 3, tRNA from C. albicans strain 304; lane 4, tRNA from C. albicans strain C9. Hb is the globin polypeptide and fl' is the fl-globin-specific, U G A readthrough polypeptide. The lysate used was prepared from a sup + [rho °] strain and thus lacked the major endogenous, mitochondrially coded U G A suppressor tRNA activity [4]. (b) BMV R N A (120 p,g/ml) was translated in vitro and t R N A s were added at a final concentration of 80 # g / m l as follows; lane 1, no tRNA; lane 2, t R N A from a sup + strain of S. cerevisiae; lane 3, tRNA from C. albicans strain 304; lane 4, tRNA from S. cerevisiae strain carrying the S U P 6 1 amber suppressor. CP = BMV coat protein; CP' and CP" are the two BMV-specific amber readthrough polypeptides [13].

28 TABLE I ABILITY OF WILD-TYPE tRNA TO TRANSLATE THE U G A A N D U A G T E R M I N A T I O N C O D O N S IN A Y E A S T CELL-FREE SYSTEM Total t R N A was isolated from s t a t i o n a r y - p h a s e cells of S. cerevisiae, S. pombe, C. albicans and E. coil, as previously described [13]. Mucor t R N A was the gift of Professor P. Sypherd (U.C. lrvine), and t R N A s from reticulocyte, soybean and wheat germ were gifts from Dr. N. Wills (University of Utah). t R N A (80 p , g / m l ) was a d d e d to a yeast cell-free system a n d r e a d t h r o u g h of the r a b b i t ,8-globin U G A t e r m i n a t o r and the Brome Mosaic Virus coat protein cistron U A G t e r m i n a t o r was assayed as previously described [17]. The cell-free system was p r e p a r e d from a sup ~ [psi + ] [rho tl] strain of S. cereetsiae to e l i m i n a t e e n d o g e n o u s UGA readthrough by the m i t o c h o n d r i a l l y - c o d e d t R N A I~p [4]. S p e r m i d i n e (0.2 raM) was present in all assays [21]. n.t. = not tested. Source o f t R N A

UGA readthrough (%)

UAG readthrough (%)

No tRNA Saccharomyces cerevisiae Schizosaccharono,eespombe Candida albicans (304) Candida albicans (C9) Escherichia coil R a b b i t reticulocytes Mucor racemosus Soybean W h e a t germ S. cerevlsiae ( S U P 6 1 - a )

< 1 37.2 4.1 51.6 44.7 18.7 2.2 8.1 < 1 < 1 n.t.

< 1 < 1 < 1 49.6 45.0 < 1 < 1