Krikor Nicoghosian

Characterization of a chemically synthesized RNA having the sequence of the yeast initiator tRNAMet

A 75‐unit long oligoribonucleotide corresponding to the sequence of the Saccharomyces cerevisiae initiator tRNA was synthesized chemically. The crude RNA was purified, and the sequence was verified by RNA sequencing techniques. A particularly useful purification step involved hydrophobic chromatography on BND‐cellulose. The purified RNA could be aminoacylated to 28% of a bona fide initiator tRNAMetsample and threonylated to 76 % of the level observed with native tRNAfMet from E. coli.

Halobacterium cutirubrum tRNA sequences

The primary structures of nine Halobacterium cutirubrum tRNAs are presented. These tRNAs are compared with other archaebacterial tRNAs and in particular with their H. volcanii counterparts. Striking similarities are observed among isoacceptor tRNA species; observed differences between a given tRNA pair of the two halophiles having the same anticodon vary from 0 to 14 nucleotides. Particular features including modified nucleotides of each of the nine tRNAs are indicated.

Glycine and asparagme tRNA sequences from the archaebacteriuin, Methanobacterium thermoautotrophicum

Two tRNA sequences from Methanobacterium thermoautotrophium are reported. Both tRNAGly GCC and tRNANUU Asn, the first tRNA sequences from methanogens, were determined by partial hydrolyses (both chemical and enzymatic) and analyzed by gel electrophoresis. The two tRNAs contain the unusual T‐loop modifications, Cm and m1I, which are present in other archaebacterial tRNAs. Finally the presence of an unknown modification in the D‐loop has been inferred by a large jump in the sequence ladder. These tRNAs are approximately equidistant from eubacterial or eukaryotic tRNAs.

Archetypical features in tRNA families.

SummaryA compilation of known tRNA, and tRNA gene sequences from archaebacteria, eubacteria, and eukaryotes permits the construction of tRNA cloverleafs which show conserved structural elements for each tRNA family. Positions conserved across the three kingdoms are thought to represent archetypical features of tRNAs which preceded the divergence of these kingdoms.

The nucleotide sequence of mannosyl-Q-containing tRNAAsp from Xenopus laevis oocytes

The nucleotide sequence of tRNAAsp from X. laevis oocytes was determined as being: (sequence in text) The tRNA is 75 nucleotides long. This sequence is very similar (75% to 97% identity) to all other eukaryotic tRNAAsp sequenced so far, except for the bovine liver tRNAAsp (32% identity). The relation between the presence of a mannosyl group on queuosine (Q) at position 34 and the nucleotide sequence of the anticodon loop is discussed.

A novel RNA digesting activity from commercial polynucleotide phosphorylase

RNase A4 is a new RNase activity found as a contaminant in commercial polynucleotide phosphorylase. This enzyme has the ability of hydrolyzing the phosphodiester bond between pyrimidine‐A in both loop and paired regions of RNA.

The use of cloned tRNA genes for the purification and measurement of specific tRNAs

We have previously reported the ability of a cloned tRNAMeti gene (pt145) to bind tRNAMeti specifically [5]. In this paper, we show that a pBR322 plasmid containing the tRNAAsn gene of Xenopus (pt38 - donated by Stuart Clarkson) will specifically bind to mouse tRNAAsn when total mouse tRNA, extracted from uninduced Friend erythroleukemia cells, is hybridized to the gene probe. One-dimensional electrophoresis of the hybridizing tRNA in 20% polyacrylamide reveals one major band and several small-molecular-weight minor bands. The hybridizing tRNA has been identified as tRNAAsn by partial RNA sequencing and the detection of both the Q base and t6A. The steady-state concentration of tRNAAsn in the uninduced Friend cell was determined by hybridizing tRNA labeled in vitro to pt38. 1% of the total tRNA hybridized, representing 0.017 pg tRNAAsn/cell. The fraction of newly synthesized tRNA representing tRNAAsn or tRNAMeti was also determined by hybridizing tRNA labeled in vivo to either pt38 or pt145, respectively. 0.96% and 0.85% of the tRNA hybridized to pt38 and pt145, respectively.