E L S E V I E R Genetic Analysis: Biomolecular Engineering

13 (1996) 45 47

ALYSIS Blomolecular Engineering

Isolation of genes encoding tRNA binding proteins by probing an expression library with unmodified tRNA

Claes Gustafsson ~'*

Department of Microbiology, University of Umed, 901 87 Umed, Sweden

Received 15 January 1996; revised 9 May 1996; accepted 10 May 1996

Abstract

A rapid method for isolating Escherichia coli genes encoding proteins which bind unmodified, but not modified, tRNA is described. The method is generally applicable, and can be used to clone many RNA binding proteins where a structured RNA ligand is known.

Keywords: 2gtl 1 Expressior. library; tRNA binding proteins; Ordered E. coli chromosomal inserts

There are some 30 different modified nucleotides present in tRNA from E. coli, many found at several locations. It has been estiLmated that about 45 different tRNA modifying enzymes are involved in modifying these nucleotides of tRNA in E. coli [1]. To date, less than half of the structural genes for these enzymes have been identified. The purpose of the present study was to establish a new procedure for cloning genes encoding tRNA modifying enzymes not easily cloned by other means. A novel way to isolate genes encoding potential tRNA modifying enzyme:s as well as other tRNA bind- ing proteins is described.

The fully modified tRNA interacts with a number of proteins such as aminoacyl-tRNA synthetases, initia- tion factors, elongation factors, ribosomal proteins and peptidyl-hydrolases. When screening a phage library with unmodified tRNA, clones containing these and other general nucleic acid binding proteins are likely to generate false positive signals. It has been shown that at least in the case of the tRNA modifying enzyme TrmA

*Corresponding author: , Tel: +415 343 8673; fax: +415 343 2931; email: claes@calvin.ucsf.edu

1present address: Kosan 13iosciences, Inc. 1450 Rollins Rd., Burlingame, CA 94010, USA

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(tRNA(mSU54) methyltransferase), the binding affinity of the enzyme to the substrate tRNA decreases 40-fold after modification (X.R. Gu and D.V. Santi, personal communications). The screening method presented here relies on this being a general trait of tRNA modifying enzymes. By selecting clones that specifically bind un- modified but not modified tRNA, mainly clones encod- ing tRNA modifying enzymes are isolated.

In the presence of Mg 2 +, unmodified tRNA is highly structured. Unlike most other RNA of the same size, it is remarkably resistant to degradation and easily syn- thesized with a high specific activity by T7 RNA poly- merase making it an ideal probe. The total overall structure changes upon modification [2], but both the unmodified and the modified form of tRNA are struc- turally rigid and stable.

Other protocols utilizing RNA probes when screen- ing expression libraries have been published [3,4], al- though these protocols rely on unstructured RNA for binding specificity and in the latter case uses UV crosslinking for detection. In neither case have the function of a cloned gene products been established. Another limitation with the previously published proto- cols is the lack of an inherent negative control, such as the fully modified tRNA used in our study, to discrim- inate between non-specific and specific RNA binders.

46 c. GustaJg'son / Genetic Analysis: Biomolecular Engineering 13 (1996) 45-47

The probe was synthesized using BstNI linearized plasmid pVAL119-21, containing the gene for tRNA va~l ligated after the T7 RNA polymerase promoter [5], as template. T7 RNA polymerase directed transcription of tRNA v"~J was performed using 32p-~UTP in the tran- scription mixture [6]. The synthesized tRNA vail was purified by agarose gel electrophoresis and eluted from the agarose using Magic prep TM (Promega, Madison, WI). The tRNA probe was incubated at 90°C for 2 min and cooled to room temperature in the presence of 10 mM MgC12 before adding it to the hybridization buffer. A 2gtl 1 genomic E. coli expression library (Cat. nr. XLl l5b , Clontech, Palo Alto, CA) was propagated in E. coli strain Y1090 following the protocol of Huynh et al. [7]. The expression library was plated at 3000 plaque forming units per Petri dish and blotted to Hybond- C TM nitrocellulose filters (Amersham, Arlington Heights, IL) presoaked in 10 mM IPTG, as previously described [8]. Each Petri dish with plaques was lifted in duplicates to nitrocellulose filter and only plaques that gave positive signal on both filters were further ana- lyzed. Doublets of filters containing the blotted 2gtl 1 plaques were pre-incubated in TDMN-50 (10 mM Tris-HC1 pH 7.5, 1 mM DTT, 10 mM MgC12, 50 mM NaC1) for 20 min. Pre-incubation buffer was replaced by hybridization buffer TDMN-50 containing 10 pg/ml calf thymus DNA and labeled tRNA (100 ng/ml, 1 × 10 6 cpm/ml). Filters were incubated at room tempera- ture for 2 h with gentle agitation, followed by 4 × 5 min washes with TDMN-50. The filters were semi- dried, and exposed on a PhosporImager screen (Molec- ular Dynamics, Sunnyvale, CA) for an appropriate time. Blotted plaques giving positive signals were iso- lated and rescreened three times until tRNA binding plaques were free from contaminating plaques and con- sistency of binding could be determined. In a first attempt to establish the system, 15000 2g t l l plaques containing on average 2 kb inserts were screened, and several plaques giving positive signal were identified (Fig. 1).

In order to separate general tRNA, or nucleic acids binding clones from clones binding only unmodified tRNA, plaques were incubated with end labeled [6] modified E. coli tRNA (Sigma, St. Louis, MO) under identical hybridization conditions as above. Only those blotted plaques that hybridized to unmodified, but not modified tRNA, were retained. Possible false positive signals that could not be discarded include proteins binding features of T7 synthesized RNA such as the 5'-triphosphate (not present in mature tRNA) or spe- cific tRNA vail binding proteins that may not be de- tected when analyzing binding using a complete collection of modified tRNAs as a second probe.

Phages from plaques giving positive signal were purified and the inserts were amplified by PCR using oligonucleotides complementary to both sides of the

Fig. 1. Northwestern primary screening of an E. coli genomic expres- sion library using unmodified tRNA va" as probe. A total of 6000 pfu were plated on a 132-mm Petri dish and screened for tRNA binding proteins as described. Encircled plaques show positive signals.

Ec oRI cloning site in ,~gtll as described in the Clon- tech protocol. The PCR amplified products were iso- lated on an agarose gel and DNA from the corresponding gel slice purified by Magic prep TM

(Promega), endlabeled by T4 polynucleotide kinase [6] and hybridized to a membrane with blotted plaques of a recombinant 2 phage library carrying ordered E. coli chromosomal inserts to determine the chromosomal location of the inserts (Fig. 2) [9]. The membranes were purchased from Takara Biochemicals (Kyoto, Japan). The hybridization and washing of the membranes were carried out according to the recommendations of the supplier. The amplified fragments were also cloned into pBluescript and sequenced using standard molecular biology protocols.

In this initial screen, three tRNA binding plaques were identified. Phage 2gtl 1-1 contains amino acid 33 to 240 in the previously sequenced orf f402 [10] located at 79.9 min on the E. coli chromosome (Kohara phage 602). Phage 2gt l l -2 contains a 1 kb insert and is located at 68.1 rain (Kohara phages 505 and 506). Phage 2gtl 1-3 contains a chromosomal insert of 2.1 kb located at 24.9 min (Kohara phages 235 and 236).

Fig. 2. Representative Southern hybridization of an amplified PCR product from a putative tRNA modifying enzyme to an ordered genomic library. Each square giving background signal is composed of six independent clones. This particular PCR amplified product (Agtl 1-1) hybridizes to Kohara phage 602, corresponding to minute 79.9 on the E. coil chromosome.

C. Gustafsson / Genetic Analysis: Biomolecular Engineering 13 (1996) 45-47 47

Acknowledgements

The work was carried out in the labora tory of G l e nn R. Bj6rk, who is grateful]ly acknowledged. Suppor t was provided by the Swedish Cancer Society (Project 680), and The Swedish Natura l Science Counci l (project BBU 2930).

References

[1] Bj6rk GR. Prog. Nucleic Acid Res Mol Biol 1995; 50:263 339.

[2] Yue D, Kintanar A, Horowitz J. Biochemistry 1994; 33: 8905-8911.

[3] Qian Z, Wilusz J. Anal Biochem 1993; 212: 547-554. [4] Werner R, Mulbach HP, Guitton MC. BioTechniques 1995;

19: 218-221. [5] Chu WC, Horowitz J. Nucleic Acids Res 1989; 17: 7241-7252. [6] Sambrook J, Fritsch EF, Maniatis T, eds. Molecular Cloning:

A Laboratory Manual. Cold Spring Harbor Laboratory Press, New York, 1989.

[7] Glover DM, ed. DNA Cloning: A Practical Approach. Vol- ume 1. IRL Press, Oxford, 1985; 49-78.

[8] Singh H, LeBowitz JH, Baldwin AS, Sharp PA. Cell 1988; 52: 415-423.

[9] Kohara Y, Akiyama K, Isono K. Cell 1987; 50: 495-508. [10] Sofia HJ, Burland V, Daniels DL, Plunkett III G, Blattner

FR. Nucleic Acids Res 1994; 22:2576 2586.