In order to understand the structure, genomic organization and expression of a multigene isoacceptor family, a human serine t RNA gene family was studied.
A human genome library has been screened using purified human serine t RNA as a probe and five different positive lambda phage clones were isolated (Yoo, 1985). Characterization of the phage DNAs revealed that six t RNA genes were contained in different restriction fragments, which suggests that most human serine t RNA genes are dispersed throughout the genome.
From the clones, four serine t RNA genes were subcloned and the nucleotide sequences have been determined in this study. The nucleotide sequence analyses revealed that all the structural genes are composed of 82 bp and do not have any intervening sequences. As in other eucaryotic t RNA genes, they do not encode the 3'-terminal CCA found in mature t RNAs and 3' flanking regions have a stretch of T residues on the noncoding strand which function as sites for termination of transcription by RNA polymerase III. The anticodon sequences of the genes are 5'-TGA-3' or 5'-AGA-3' that are expected to recognize serine codons UCN. Moreover, the sequences of the noncoding strand of the genes form typical cloverleaf conformations with all the appropriate conserved and semiconserved bases.
Interstingly, 200 bp segment of two t RNA (AGA) genes encompassing 5' flanking regions and the coding regions are identical without single base change, demonstrating that the two genes arose by gene duplication event(s).
The coding sequences of the genes are highly homologous. The minor sequence variations occurred only at 5' terminal nucleotide of anticodon sequence, extra arm loop, acceptor stem and T stem without change in conserved and semiconserved bases that function as an internal split promoter. However, the 5' and 3' flanking regions do not show any significant sequence homology. In addition, two t RNA (AGA) genes are identical to rat liver serine t RNA (IGA), which suggests that mammalian serine t RNA (NGA) genes are highly conserved.
All the t RNA genes were transcribed by RNA polymerase III in an in vitro transcription reaction using He La nuclear extract, and the primary transcripts were partially processed to mature t RNAs. The result demonstrates that the human t RNA genes could be functional in vivo.
