In Vivo Deuteration of Transfer RNAs: Overexpression and Large-Scale Purification of Deuterated Specific tRNAs

Ralf Jnemann 2 Jrg Wadzack 2 Francisco J. Triana-Alonso 1 2 Jrg-Uwe Bittner 2 Jol Caillet 0 Thierry Meinnel 4 Kalju Vanatalu 3 Knud H. Nierhaus 2 0 Laboratoire de Biochimie , Ecole Polytechnique, 91128 Palaiseau cedex, France 1 Centro de Investigaciones Biomedicas, Universidad de Carabobo , Maracay, Venezuela 2 Max-Planck-Institut fr Molekulare Genetik , Ihnestrae 73, 14195 Berlin-Dahlem, Germany 3 Institute of Chemical Physics and Biophysics , Akadeemia tee 23, 0026 Tallin, Estonia 4 Institut de Biologie Physico-Chimique , 13 rue Pierre et Marie Curie, 75005 Paris, France Structural investigations of tRNA complexes using NMR or neutron scattering often require deuterated specific tRNAs. Those tRNAs are needed in large quantities and in highly purified and biologically active form. Fully deuterated tRNAs can be prepared from cells grown in deuterated minimal medium, but tRNA content under this conditions is low, due to regulation of tRNA biosynthesis in response to the slow growth of cells. Here we describe the large-scale preparation of two deuterated tRNA species, namely DtRNAPhe and DtRNAfMet (the method is also applicable for other tRNAs). Using overexpression constructs, the yield of specific deuterated tRNAs is improved by a factor of two to ten, depending on the tRNA and growth condition tested. The tRNAs are purified using a combination of classical chromatography on an anion exchange DEAE column with reversed phase preparative HPLC. Purification yields nearly homogenous deuterated tRNAs with a chargeability of ~ 1400-1500 pmol amino acid/A260 unit. The deuterated tRNAs are of excellent biological activity. - For structural investigations of biological molecules which function as part of multi-component systems, electron microscopy, X-ray diffraction, neutron scattering and NMR techniques are the most important direct physical methods that allow studies of structurefunction relationships (1). Information from these methods is an indispensable prerequisite for the incorporation of the details of sequence-based data into consistent models. The great advantage of neutron scattering is that it can be used for very large molecules or multi-subunit complexes which can be analysed in solution, thus retaining the functional conformation. * To whom correspondence should be addressed One central molecule of the translational apparatus is tRNA, with its various complexes (2). The three-dimensional structure of tRNAs was solved for tRNAPhe more than 20 years ago (3,4) and appears to be in general the same for all tRNA species. Nevertheless, the tRNA-containing complexes formed during protein synthesis are still the subject of intense structural investigation. In the last decade the structures of some tRNA complexes with their specific aminoacyl synthetase (aaRS) have been solved at atomic resolution by X-ray diffraction (reviewed in 5). In addition, the interaction between the tRNA and aaRS can be understood in detail by dynamic studies using various NMR techniques (6). Recently the ternary complex elongation factor TuGTPtRNA was crystallized successfully and the structure has been solved at atomic resolution (7). The situation for structural investigation of ribosome complexes is more difficult, because it is a multi-component ribonucleoprotein particle (57 components in Escherichia coli 70S ribosomes) with a mass of ~ 2300 kDa. To investigate such a large particle most of the structural methods cannot be easily applied. Crystallization and X-ray diffraction would probably lead to a detailed structural model, but crystallization of functional complexes is quite laborious and the phase problem is difficult to solve. Therefore, structures derived from X-ray diffraction of crystals will not be available for many years. At present only neutron scattering techniques (8) are capable of yielding a medium resolution overall structure of the tRNAribosome complex (9) by a direct physical method. Unfortunately, NMR and neutron scattering techniques need partially or even fully deuterated compounds in large quantities. However, the production of deuterated molecules is expensive and often high biological activity cannot be achieved easily, since cells grow only slowly in deuterated medium, resulting in low yield and severely reduced activity. Due to the growth rate regulation of tRNAs (10,11) the yield of tRNA is dramatically reduced when prepared from cells grown in deuterated medium. Recently a cultivation method has been described which allows the preparation of E.coli cells in kilogram quantities with high biological activity and almost 100% deuteration (12). Here we combine this method with the use of overexpression systems for specific tRNAs to increase the yield of fully deuterated tRNAs by a factor of up to 10. In addition, we describe a large-scale method to purify these tRNAs to near homogeneity preserving full biological activity. MATERIALS AND METHODS Chemicals and bacterial strains Radioactively labelled amino acids were purchased from Amersham-Buchler (Braunschweig, Germany) and restriction enzymes from New England Biolabs (Beverly, MA). All other chemicals were pro analysi grade and purchased from Merck (Darmstadt, Germany). Escherichia coli MRE600rif (12) is a strain which: (i) is adaptable to growth on deuterated media (13); (ii) contains low levels of ribonuclease I activity (14); and (iii) tolerates high doses of rifampicin. This strain was used for all cultivations in deuterated media. As a reference strain HB101 (15) containing the same plasmids as the MRE600rif derivatives was grown in protonated LB medium (see below). The plasmid pPhe was previously described as pPP15 (16). It is a pBR322 derivative containing the pheV gene, which codes for tRNAPhe, under the control of the natural P2 promoter, which is the second of a tandem promoter pair. Plasmid pMet (17) was a kind gift of U. RajBhandary. It carries the gene for tRNAfMet behind the natural promoter. The E.coli tRNAfMet gene cloned into the plasmid plppMet (previously described as pBStRNAMetfY; 18) is under the control of a synthetic lipoprotein promoter lpp (19) and has several modifications at the level of the 5 maturation sequence allowing maturation by RNase P in vivo. The tRNA transcription region is terminated by the strong terminator of the rrnC operon. This construct allows very high levels of overexpression. Plasmids pBR322 (Boehringer, Mannheim, Germany) and pBluescript (Stratagene, La Jolla, CA) were used for cloning purposes and as control plasmids (minus tRNA gene). Cloning of tRNA genes Plasmids pPhe, pMet and plppMet were used directly for transformation of HB101 or MRE600rif respectively by electroporation using a BioRad gene pulser. Plasmid plppMetPhe was constructed by subsequent cloning of the PstIHpaI fragment of pPhe (the fragment contains the tRNAPhe gene) and the XhoIHindIII fragment of plppMet (containing the cassette lpp promotertRNAfMet generrnC terminator) into the respe (...truncated)