Effects of 5′ flanking sequences and changes in the 5′ internal control region on the transcription of rice tRNA^Gly_GCC gene

Abstract

A stretch of 71 nucleotides in a 1.2 kilobase pair Pst I fragment of rice DNA was identified as tRNA^{Gl y}_GCC gene by hybridization and nucleotide sequence analyses. The hybridization of genomic DNA with the tRNA gene showed that there are about 10 glycine tRNA genes per diploid rice genome. The 3′ and 5′ internal control regions, where RNA polymerase III and transcription factors bind, were found to be present in the coding sequence. The gene was transcribed into a 4S product in an yeast cell-free extract. The substitution of 5′ internal control region with analogous sequences from either M13mp19 or M13mp18 DNA did not affect the transcription of the gene in vitro. The changes in three highly conserved nucleotides in the consensus 5′ internal control region (RGYNNARYGG; R = purine, Y = pyrimidine, N = any nucleotide) did not affect transcription showing that these nucleotides are not essential for promotion of transcription. There were two 16 base pair repeats, 'TGTTTGTTTCAGCTTA' at -130 and -375 positions upstream from the start of the gene. Deletion of 5prime flanking sequences including the 16 base pair repeat at -375 showed increased transcription indicating that these sequences negatively modulate the expression of the gene.