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The genes involved in the 2,3-butanediol pathway coding for ct-acetolactate decarboxylase, a-acetolactate synthase (ae-ALS), and acetoin (diacetyl) reductase were isolated from Klebsiela terrigena and shown to be located in one operon. This operon was also shown to exist in Enterobacter aerogenes. The budAl gene, coding for ot-acetolactate decarboxylase, gives in both organisms a protein of 259 amino acids. The amino acid similarity between these proteins is 87%. The K. terrgena genes budB and budC, coding for at-ALS and acetoin reductase, respectively, were sequenced. The 559-amino-acid-long cx-ALS enzyme shows similarities to the large subunits of the Escherichia coli anabolic a-ALS enzymes encoded by the genes ilvB, ilvG, and ilvl. The K. terrigena a-ALS is also shown to complement an anabolic ce-ALS-deficient E. coli strain for valine synthesis. The 243-amino-acid-long acetoin reductase has the consensus amino acid sequence for the insect-type alcohol dehydrogenase/ribitol dehydrogenase family and has extensive similarities with the N-terminal and internal regions of three known dehydrogenases and one oxidoreductase.

This paper presents the first example of a complete gene sequence coding for and expressing a biologically functional human tRNA methyltransferase: the hTRM1 gene product tRNA(m(2)(2)G)dimethyltransferase. We isolated a human cDNA (1980 bp) made from placental mRNA coding for the full-length (659 amino acids) human TRM1 polypeptide. The sequence was fairly similar to Saccharomyces cerevisiae Trm1p, to Caenorhabditis elegans TRM1p and to open reading frames (ORFs) found in mouse and a plant (Arabidopsis thaliana) DNA. The human TRM1 gene was expressed at low temperature in Escherichia coli as a functional recombinant protein, able to catalyze the formation of dimethylguanosine in E.coli tRNA in vivo. It targeted solely position G(26) in T7 transcribed spliced and unspliced human tRNA(Tyr) in vitro and in yeast trm1 mutant tRNA. Thus, the human TRM1 protein is a tRNA(m(2)(2)G(26))dimethyltransferase. Compared with yeast Trm1p, hTRM1p has a C-terminal protrusion of approximately 90 amino acids which shows similarities to a mouse protein related to RNA splicing. A deletion of these 90 C-terminal amino acids left the modification activity in vitro intact. Among point mutations in the hTRM1 gene, only those located in conserved regions of hTRM1p completely eliminated modification activity.

N2,N2-dimethylguanosine (m2(2)G) is a characteristic nucleoside that is found in the bend between the dihydro-uridine (D) stem and the anticodon (AC) stem in over 80% of the eukaryotic tRNA species having guanosine at position 26 (G26). However, since a few eukaryotic tRNAs have an unmodified G in that position, G26 is a necessary but not a sufficient condition for dimethylation. In yeast tRNA(Asp) G26 is unmodified. We have successively changed the near surroundings of G26 in this tRNA until G26 became modified to m2(2)G by a tRNA(m2(2)G26)methyltransferase in Xenopus laevis oocytes. In this way we have identified the two D-stem basepairs C11-G24, G10-C25 immediately preceding G26 as major identity elements for the dimethylating enzyme modifying G26. Furthermore, increasing the extra loop in tRNA(Asp) from four to the more usual five bases influenced the global structure of the tRNA such that the m2(2)G26 formation was drastically decreased even if the near region of G26 had the two consensus basepairs. We conclude that not only are the two consensus base pairs in the D-stem a prerequisite for G26 modification, but also is any part of the tRNA molecule that influence the 3D-structure important for the recognition between nuclear coded tRNAs and the tRNA(m2(2)G26)methyltransferase.

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