The tRNASer-isoacceptors and their genes in Nicotiana rustica: genome organization, expression in vitro and sequence analyses

Abstract

The existence of six serine codons results in a complex pattern of tRNA(Ser) isoacceptors in organisms and organelles. According to the original wobble hypothesis, a minimum of three isoacceptors should be sufficient to read the six serine codons. We have isolated five cytoplasmic tRNAs(Ser) from leaves of Nicotiana rustica. Their nucleotide sequences identify them as four different isoacceptors with the anticodons cm5UGA, CGA, IGA and GCU. For tRNA(Ser) with IGA anticodon, two species have been detected which vary only by one nucleotide in the long extra arm. The first three isoacceptors recognize codons of the type UCN whereas the fourth isoacceptor reads the two serine codons AGC and AGU. The tRNA(Ser) sequences were used to design appropriate primers for the amplification of Nicotiana nuclear tRNA(Ser) genes by the polymerase chain reaction (PCR). A total number of eight tRNA(Ser) genes differing in the coding region were thus identified. Selected PCR DNA fragments were then employed as probes for the isolation of the corresponding genes from a nuclear DNA library of N. rustica. Sequence analyses revealed that five of the isolated seven clones contained tRNA(Ser) genes which are identical in sequence with the five cytoplasmic tRNAs(Ser) mentioned above. None of them contains an intervening sequence. This is the first time that all putative cellular tRNA(Ser) isoacceptors and their corresponding genes have been characterized in an eukaryotic organism. Most of the tRNA(Ser) genes are functional as deduced from in vitro transcription and processing studies. Two of the genes yield pre-tRNAs(Ser) which are not or poorly converted to mature tRNA in a plant extract. The approximate tRNA(Ser) gene number was estimated by hybridization of specific DNA probes to Eco RI-cleaved Nicotiana nuclear DNA. The overall hybridization pattern indicates that members of each particular tRNA(Ser) gene family do not appear to be clustered but distributed randomly throughout the Nicotiana genome.

Partial Purification and Characterization of Nuclear Ribonuclease P from Wheat

“…An intact pre-tRNA tertiary structure is required for clevage by wheat germ RNase P. A number of nuclear plant tRNA gene variants have been described which are defective in maturation of their transcripts when assayed in an unfractionated wheat germ processing system (Stange et al, 1991;Fuchs et al, 1992;Teichmann et al, 1994). We have used some of these naturally occurring mutants to determine the influence of the implied structural alterations in the corresponding pre-tRNAs on cleavage by purified wheat nuclear RNase P. Tn contrast to the Escherichiu coli enzyme, which recognizes a variety of substrates diverging from the canonical tRNA structure Guerrier-Takada et al, 1988;Forster and Altman, 1990), the wheat enzyme is very sensitive against structural aberrations: changes involving the T-stem and T-loop domain, and even the presence of an intron in the anticodon loop, significantly reduce the cleavage efficiency.…”

“…Stange and Beier, 1987). Similarly, although investigation of a variety of pre-tRNAs indicated that mutations disrupting the tRNA structure severely impair overall processing in this plant in vitro system (Stange et al, 1991 ;Fuchs et al, 1992;Teichmann et al, 1994), it is not clear at present which of the enzymes involved is most sensitive to these changes. To approach these questions, and to gain insight into the molecular composition and substrate requirements of nuclear RNase P from higher as percentage product formation.…”

“…Some naturally occurring tRNA variant genes were available as genomic clones (Fuchs et a]., 1992;Stange et al, 1991;Teichmann et al, 1994). PCR was used to put the genes under control of the T7 promoter and to add a FokI restriction site for generation of transcripts terminating at U74.…”

Pre‐tRNA 3′‐Processing in Saccharomyces cerevisiae

“…Plasmids pAtSl, pAtS5 and pAtS6 coding for functional (pAtS6) and nonfunctional (pAtS 1 and pAtS5) Arubidopsis thalianck pre-tRNAs"' have been described by Bcier and Beier (1992). Plasmids pNtS3, pNtS4, pNtS5 and pNtS7 coding for functional (pNtS3, pNtS5 and pNtS7) and non-functional (pNtS4) Nicotiana rusticu pretRNAs'" have been described by Teichmann et al (1994). Plasmid pPMS coding for an intron-containing functional pretRNA"" from S. cerevisiue has been described by Olson et al (1981).…”

The tRNATyr multigene family of Triticum aestivum: genome organization, sequence analyses and maturation of intron‐containing pre‐tRNAs in wheat germ extract

“…We have shown that most plant tRNA genes are efficiently transcribed in both extracts [13,25,28]. Here we have performed in vitro transcription of pTtY4 to pTtY9 in HeLa cell nuclear extract at 1 mM Mg 2+ concentration.…”