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The 2 -5 RNA Ligase of Escherichia Coli

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The 2 -5 RNA Ligase of Escherichia Coli View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Caltech Authors - Main THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 271, No. 49, Issue of December 6, pp. 31145–31153, 1996 © 1996 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. The 2*-5* RNA Ligase of Escherichia coli PURIFICATION, CLONING, AND GENOMIC DISRUPTION* (Received for publication, August 7, 1996, and in revised form, September 18, 1996) Eric A. Arn‡ and John N. Abelson From the Division of Biology 147-75, California Institute of Technology, Pasadena, California 91125 An RNA ligase previously detected in extracts of Esch- editing in kinetoplastids (5). At least two other species of RNA erichia coli is capable of joining Saccharomyces cerevi- ligase, the metazoan-specific “animal pathway” RNA ligase (6) siae tRNA splicing intermediates in the absence of ATP and the archaeal stable RNA splicing ligase (7), have been to form a 2*-5* phosphodiester linkage (Greer, C., Javor, identified, but the precise in vivo substrate(s) and function(s) of B., and Abelson, J. (1983) Cell 33, 899–906). This enzyme these enzymes have yet to be determined. specifically ligates tRNA half-molecules containing nu- The existence of RNA ligase in bacteria in the absence of cleoside base modifications and shows a preference bacteriophage infection was discovered in this laboratory in among different tRNA species. In order to investigate 1983 (8). An activity capable of performing the ligation step of the function of this enzyme in RNA metabolism, the ligase was purified to homogeneity from E. coli lysate eukaryotic tRNA splicing was detected in extracts of a wide utilizing chromatographic techniques and separation of range of bacteria including members of the Alpha and Gamma proteins by SDS-polyacrylamide gel electrophoresis. A subdivisions of proteobacteria, green sulfur bacteria, and low G single polypeptide of approximately 20 kilodaltons ex- 1 C content Gram-positive bacteria (8). In extracts of E. coli hibited RNA ligase activity. The amino terminus of this the ligase activity had a substrate specificity restricted to 4 of protein was sequenced, and the open reading frame the 10 Saccharomyces cerevisiae tRNA splicing intermediates: (ORF) encoding it was identified by a data base search. tRNA Tyr, Phe, Lys2, and Trp half-molecules. The reaction This ORF, which encodes a novel protein with a pre- mechanism of the E. coli RNA ligase apparently differed from dicted molecular mass of 19.9 kDa, was amplified from E. that of known RNA ligases since it did not require a nucleoside coli genomic DNA and cloned. ORFs coding for highly triphosphate cofactor, and the product contained an unusual similar proteins were detected in Methanococcus jan- 29-59 phosphodiester bond at the ligated junction (8). naschii and Bacillus stearothermophilus. The chromo- The discovery of RNA ligase in E. coli implies the existence of somal gene encoding RNA ligase in E. coli was dis- rupted, abolishing ligase activity in cell lysates. Cells a novel form of bacterial RNA processing. No intervening se- lacking ligase activity grew normally under laboratory quences of the type found in eukaryotic nuclear or archaeal conditions. However, moderate overexpression of the tRNA genes (which require enzymatic excision and religation) ligase protein led to slower growth rates and a temper- occur in known bacterial tRNA genes, although self-splicing ature-sensitive phenotype in both wild-type and RNA introns are found in the tRNA genes of certain cyanobacteria ligase knockout strains. The RNA ligase reaction was (9) and some proteobacteria (10). In fact, no introns of any kind studied in vitro using purified enzyme and was found to are found in the full genomic complement of tRNA genes in E. be reversible, indicating that this enzyme may perform coli, Mycoplasma capricolum, and Hemophilus influenzae (11– cleavage or ligation in vivo. 13). To date no RNA processing event that would require the action of an RNA ligase enzyme has been observed to occur in any bacteria, indicating that elucidation of the substrate and RNA ligases have been detected in organisms throughout all function of the 29-59 RNA ligase should reveal a previously major divisions of the phylogenetic spectrum. Specific meta- unknown step in bacterial RNA metabolism. bolic functions, however, have been assigned to few of these A genuine in vivo function for the E. coli RNA ligase activity enzymes. T4 RNA ligase, for example, repairs nicks introduced observed in vitro is suggested by the occurrence of 29-59 link- into the anticodon loops of tRNAs in phage-restrictive Esche- ages in native E. coli RNA. Several forms of 2 -5 -linked oli- richia coli strains upon T4 infection (1); the eukaryotic tRNA 9 9 splicing RNA ligase joins tRNA half-molecules resulting from goadenylates have been detected in acid-soluble extracts of E. endonucleolytic removal of introns from eukaryotic tRNA pre- coli (14). The most abundant species of these oligoribonucleo- cursors (2–4), and an RNA ligase is apparently responsible for tides observed was 29-59-linked adenosine dinucleotide 39- the joining of guide RNA molecules to mRNA during RNA monophosphate, which was estimated to exist at an intracellu- lar concentration over 100 nM. This dinucleotide could not be an intermediate in oligoadenylate synthesis, and it has been sug- * This work was supported by a grant from the American Cancer gested that this species may be a degradation product of RNAs Society and a National Research Service Award (to E. A. A.). The costs of publication of this article were defrayed in part by the payment of containing individual 29-59 bonds among standard 39-59 link- page charges. This article must therefore be hereby marked “advertise- ages (14). Since the formation of 29-59 linkages is not catalyzed ment” in accordance with 18 U.S.C. Section 1734 solely to indicate this by RNA polymerase, these bonds must be added in a post- fact. The nucleotide sequence(s) reported in this paper has been submitted transcriptional processing event by an enzyme such as the 29-59 to the GenBankTM/EBI Data Bank with accession number(s) D26562 RNA ligase. and Swissprot P37025. In order to study the function and mechanism of the 29-59 ‡ To whom correspondence should be addressed. Current address: RNA ligase, the enzyme was purified to homogeneity from E. Dept. of Cell Biology, Harvard Medical School, 240 Longwood Ave., Boston, MA 02115. Tel.: 617-432-3819; Fax: 617-432-1144; E-mail: coli extracts. A single polypeptide was found to contain RNA [email protected]. ligase activity. This protein was partially sequenced, and the This paper is available on line at http://www-jbc.stanford.edu/jbc/ 31145 31146 Purification and Cloning of E. coli 29-59 RNA Ligase ORF1 encoding it was identified. This ORF was amplified from batchwise with 500 ml of DEAE-Sepharose CL-6B (equilibrated in EB E. coli genomic DNA and cloned. The chromosomal locus con- 1 125 mM KCl) for 30 min on ice. All subsequent steps were performed taining the ligase gene was disrupted, abolishing ligase activity at 4 °C. The DEAE/extract slurry was poured into a column, and the DEAE effluent was loaded at approximately 1 ml/min onto a 200-ml bed in cellular extracts. Cells completely lacking ligase activity of cellulose phosphate P11 (Whatman) equilibrated in EB 1 125 mM grow similarly to the parent strain. E. coli strains overexpress- KCl. ing recombinant 29-59 RNA ligase are temperature-sensitive for The phosphocellulose column was washed extensively with EB 1 500 growth. The ligase reaction was also studied using purified mM KCl and eluted with a linear KCl gradient from 0.5 to 1.5 M in EB. enzyme and was found to be reversible in vitro. Peak ligase activity fractions were pooled, diluted to 150 mM KCl, and loaded onto 25 ml of heparin Hyper-D (Biosepra) column. Ligase activ- EXPERIMENTAL PROCEDURES ity was eluted from the heparin column with a 150–700 mM KCl Strains—E. coli strains utilized were HB101, XL1-Blue (Novagen), gradient in EB, and peak fractions were pooled. The heparin pool was diluted to 150 mM KCl in EB and loaded onto a 15-ml bed column of E. and HSD947, DH10B, RecA1. Preparation of RNA Substrates for Ligation—RNA polymerase III coli tRNA linked to a Sepharose support. tRNA Sepharose (3 mg of RNA transcription of pre-tRNATyr in yeast extract was performed by the per ml of gel) was prepared as described by Rauhut et al. (19) but using method of Evans and Engelke (15), using extracts prepared as de- E. coli tRNA (Sigma) instead of yeast tRNA. Ligase was eluted from this scribed. The template for Pol III transcription of pre-tRNATyr was column using a gradient from 150 mM to 1 M KCl in EB, followed by a supercoiled pYSUP6 (16). T7 RNA polymerase transcription was per- 2 M KCl wash. The ligase eluted in a broad peak beginning around 400 formed as described by Sampson and Saks (17). pre-tRNAPhe was tran- mM KCl, trailing into the 2 M wash. scribed from the artificial pre-tRNA gene of Reyes cloned into pUC13 Active fractions were pooled, diluted to 150 mM KCl, loaded onto a (18). The template for pre-tRNATyr T7 transcription was created by 2-ml column of heparin hyper-D, and eluted with EB 1 600 mM KCl. PCR amplification from pYSUP6, adding a canonical T7 promoter at the This concentrated activity pool was then passed through a 115-ml bed of Superdex 75 gel filtration medium (Pharmacia Biotech Inc.) at a flow 59 end and a BstNI restriction site at the 39 end of the pre-tRNA, as well 1 2 87 M KCl in as altering the acceptor stem base pairs: C 3 G, T 3 G, A 3 C, and rate of 0.04 ml/min.
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