Archaeosine is a novel derivative of 7-deazaguanosine found in transfer RNAs of most organisms exclusively in the archaeal phylogenetic lineage and is present in the D-loop at position 15. We show that this modification is formed by a posttranscriptional base replacement reaction, catalyzed by a new tRNA-guanine transglycosylase (TGT), which has been isolated from Haloferax volcanii and purified nearly to homogeneity. The molecular weight of the enzyme was estimated to be 78 kDa by SDS-gel electrophoresis. The enzyme can insert free 7-cyano-7-deazaguanine (preQ0 base) in vitro at position 15 of an H. volcanii tRNA T7 transcript, replacing the guanine originally located at that position without breakage of the phosphodiester backbone. Since archaeosine base and 7-aminomethyl-7-deazaguanine (preQ1 base) were not incorporated into tRNA by this enzyme, preQ0 base appears to be the actual substrate for the TGT of H. volcanii, a conclusion supported by characterization of preQ0 base in an acid-soluble extract of H. volcanii cells. Thus, this novel TGT in H. volcanii is a key enzyme for the biosynthetic pathway leading to archaeosine in archaeal tRNAs.
Using labeled transcripts generated in vitro from squid total genomic DNA as a probe, we isolated and characterized a SINE that is present In the squid genome. The squid SINE appears to be derived from a tRNALYS. When the consensus sequences offive different SINEs with a tRNALY-like structure from distantly related species, including squid, were aligned, we found in the tRNA-unrelated region two sequence motifs that were almost identical among these five SINEs. This observation suggests a common evolutionary origin for these SINEs and/or some function(s) for these motifs. Similar sequences were unexpectedly found to be present in sequences complementary to the US regions of several mammalian retroviruses whose primer is a tRNALys. On the basis of these findings, we present a model for the generation of SINEs. We propose that they are derived from a "strong-stop DNA" with a primer tRNALys that is an intermediate in the reverse tanmscription of certain retroviruses. Our model suggests that a certain group of SINEs may have been generated by horizontal transmission, although it is not clear whether information was transmitted via a similar retrovirus or via an RNA or DNA of a SINE.
Two lysine isoacceptor tRNAs corresponding to the codons AAA and AAG, respectively, were isolated from squid (Loligo bleekeri), and their nucleotide sequences were determined. During this analysis, we discovered that the tRNA with the anticodon CUU was efficiently cleaved at a specific site in the presence of magnesium ions, whereas the tRNA with the anticodon UUU was not. Cleavage occurred almost exclusively at the phosphodiester linkage between G15 and D16 (p16). The most remarkable feature of this cleavage reaction is that the end product was not a 2',3'-cyclic phosphate but was mainly a 3'-phosphate. Thus, this reaction was distinct from the well characterized cleavage of yeast tRNA(Phe) by lead and from reactions catalyzed by various other metalloribozymes. The presence of a cytidine residue at position 60 was required for efficient cleavage but was not crucial for the reaction, and the entire tRNA molecule had to be intact for this specific and efficient cleavage reaction. The present study provides evidence that there exists a new catalytic mechanism for cleavage of tRNA that exploits biologically ubiquitous ions rather than toxic, nonessential ions such as lead.
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