The context of transcription start site regions is crucial for transcription of a plant tRNALys(UUU) gene group both in vitro and in vivo

Nuclear tRNA genes are transcribed by RNA polymerase III. The A- and B-boxes located within the transcribed regions are essential promoter elements for nuclear tRNA gene transcription. The Arabidopsis genome contains ten annotated genes encoding identical tRNALys(UUU) molecules, which are scattered on the five chromosomes. In this study, we prepared ten tDNA constructs including each of the tRNALys(UUU) coding sequences with their individual 5′ and 3′ flanking sequences, and assayed tRNA expression using an in vitro RNA polymerase III-dependent transcription system. Transcription levels differed significantly among the ten genes and two of the tRNA genes were transcribed at a very low level, despite possessing A- and B-boxes identical to those of the other tRNA genes. To examine whether the in vitro results were reproducible in vivo, the 5′ flanking sequence of an amber suppressor tRNA gene was then replaced with those of the ten tRNALys genes. An in vivo experiment based on an amber suppressor tRNA that mediates suppression of a premature amber codon in a β-glucuronidase (GUS) reporter gene in plant tissues generated nearly identical results to those obtained in vitro. Analysis of mutated versions of the amber suppressor tRNA gene, which contained base substitutions around the transcription start site (TSS), showed that the context around the transcription start sites is a crucial determinant for transcription of plant tRNALys(UUU) both in vitro and in vivo. The above transcription regulation by context around TSS differed between tRNA genes and other Pol III-dependent genes.

► In plant, TSS contexts of tRNA gene regulate transcription levels.
► Above findings were done by in vitro and in vivo.
► In vivo confirmation was the first evidence.
► GT nucleotides on TSS region reduce tRNA transcription, but not in other small RNAs.
► TCAACA motif has TSS convergence effect both in tRNA and other small RNAs genes.