Nucleotide Context Can Modulate Promoter Strength in Genes Transcribed by RNA Polymerase III.

The small nuclear RNAs 4.5SH and 4.5SI were characterized only in mouse-like rodents; their genes originate from 7SL RNA and tRNA, respectively. Similar to many genes transcribed by RNA polymerase III (pol III), the genes of 4.5SH and 4.5SI RNAs include boxes A and B, forming an intergenic pol III-directed promoter. In addition, their 5'-flanking sequences have TATA-like boxes at position -31/-24, also required for efficient transcription. The patterns of the three boxes notably differ in the 4.5SH and 4.5SI RNA genes. The A, B, and TATA-like boxes were replaced in the 4.5SH RNA gene with the corresponding boxes in the 4.5SI RNA gene to evaluate their effect on the transcription of transfected constructs in HeLa cells. Simultaneous replacement of all three boxes decreased the transcription level by 40%, which indicates decreased promoter activity in a foreign gene. We developed a new approach to compare the promoter strength based on the competition of two co-transfected gene constructs when the proportion between the constructs modulates their relative activity. This method demonstrated that the promoter activity of 4.5SI is 12 times that of 4.5SH. Unexpectedly, the replacement of all three boxes of the weak 4.5SH promoter with those of the strong 4.5SI gene significantly reduced, rather than enhanced, the promoter activity. Thus, the strength of a pol III-directed promoter can depend on the nucleotide environment of the gene.

TATA-Like Boxes in RNA Polymerase III Promoters: Requirements for Nucleotide Sequences.

tRNA and some other non-coding RNA genes are transcribed by RNA polymerase III (pol III), due to the presence of intragenic promoter, consisting of boxes A and B spaced by 30-40 bp. Such pol III promoters, called type 2, are also intrinsic to Short Interspersed Elements (SINEs). The contribution of 5'-flanking sequences to the transcription efficiency of genes containing type 2 promoters is still studied insufficiently. Here, we studied this issue, focusing on the genes of two small non-coding RNAs (4.5SH and 4.5SI), as well as B1 and B2 SINEs from the mouse genome. We found that the regions from position -31 to -24 may significantly influence the transcription of genes and SINEs. We studied the influence of nucleotide substitutions in these sites, representing TATA-like boxes, on transcription of 4.5SH and 4.5SI RNA genes. As a rule, the substitutions of A and T to G or C reduced the transcription level, although the replacement of C with A also lowered it. In 4.5SH gene, five distal nucleotides of -31/-24 box (TTCAAGTA) appeared to be the most important, while in the box -31/-24 of 4.5SI gene (CTACATGA), all nucleotides, except for the first one, contributed significantly to the transcription efficiency. Random sequences occurring at positions -31/-24 upstream of SINE copies integrated into genome, promoted their transcription with different efficacy. In the 5'-flanking sequences of 4.5SH and 4.5SI RNA genes, the recognition sites of CREB, C/EBP, and Sp1 factors were found, and their deletion decreased the transcription.

Influence of 5'-flanking sequence on 4.5SI RNA gene transcription by RNA polymerase III.

Short nuclear 4.5SI RNA can be found in three related rodent families. Its function remains unknown. The genes of 4.5SI RNA contain an internal promoter of RNA polymerase III composed of the boxes A and B. Here, the effect of the sequence immediately upstream of the mouse 4.5SI RNA gene on its transcription was studied. The gene with deletions and substitutions in the 5'-flanking sequence was used to transfect HeLa cells and its transcriptional activity was evaluated from the cellular level of 4.5SI RNA. Single-nucleotide substitutions in the region adjacent to the transcription start site (positions -2 to -8) decreased the expression activity of the gene down to 40%-60% of the control. The substitution of the conserved pentanucleotide AGAAT (positions -14 to -18) could either decrease (43%-56%) or increase (134%) the gene expression. A TATA-like box (TACATGA) was found at positions -24 to -30 of the 4.5SI RNA gene. Its replacement with a polylinker fragment of the vector did not decrease the transcription level, while its replacement with a GC-rich sequence almost completely (down to 2%-5%) suppressed the transcription of the 4.5SI RNA gene. The effect of plasmid sequences bordering the gene on its transcription by RNA polymerase III is discussed.