In vitro analysis of the sequences required for transcription of the Arabidopsis thaliana 5S rRNA genes

In vivo, we have already shown that only two of the 5S rDNA array blocks of the Arabidopsis thaliana genome produce the mature 5S rRNAs. Deletions and point mutations were introduced in an Arabidopsis 5S rDNA-transcribed region and its 5'- and 3'-flanks in order to analyse their effects on transcription activity. In vitro transcription revealed different transcription control regions. One control region essential for transcription initiation was identified in the 5'-flanking sequence. The major sequence determinants were a TATA-like motif (-28 to -23), a GC dinucleotide (-12 to -11), a 3-bp AT-rich region (-4 to -2) and a C residue at -1. They are important for both accurate transcription initiation and transcription efficiency. Transcription level was regulated by polymerase III (Pol III) re-initiation rate as in tRNA genes in which TATA-like motif is involved. Active 5S rDNA transcription additionally required an intragenic promoter composed of an A-box, an Intermediate Element (IE) and a C-box. Double-stranded oligonucleotides corresponding to different fragments of the transcribed region, used as competitors, revealed the main importance of internal promoter elements. A stretch of four T is sufficient for transcription termination. Transcription of Arabidopsis 5S rDNA requires 30 bp of 5'-flanking region, a promoter internal to the transcribed region, and a stretch of T for transcription termination.