Xenopus ribosomal gene enhancers function when inserted inside the gene they enhance

rRNA transcription in Xenopus laevis terminates near a 7-base-pair (bp) conserved sequence (T3 box) located 200 bp upstream of the site of transcription initiation for the adjacent gene promoter. We present evidence here that a 12-bp element containing the T3 box is an essential part of the terminator. Using an oocyte injection assay, we found that the 12-bp element (but not the T3 box alone) severely reduced the amount of RNA detectable at sites downstream from itself and that the T3 box within the 12-bp element was required to specify the formation of correct 3' ends. This requirement for the 12-bp element was also seen in pulse-label experiments by using a homogenate of oocyte nuclei, but the present data did not allow us to determine the exact mechanism by which the 12-bp element acts. Removal of the T3 region from its normal location allowed a significant amount of readthrough transcripts to accumulate, indicating that additional sequences may be required for complete terminator function.In Xenopus laevis oocytes, the entire ribosomal gene repeat unit is transcribed, except for about 200 base pairs (bp) upstream of the transcription start site (2, 10). As is diagrammed in Fig. 1, synthesis of a 7.8-kilobase-long precursor starts at the gene promoter, and its 3' end is formed by a type of RNA processing at a site we designated T2. Transcription continues beyond T2, but in oocytes at least, the RNA is very unstable. Synthesis of this unstable RNA continues across the intergenic spacer until site T3 is reached 215 bp upstream of the transcription initiation site of the next gene. Nuclear runoff assays clearly suggest that T3 is a termination site, since no transcription was detected between T3 and the adjacent transcription initiation site. Throughout this paper we refer to events at T3 as termination, although the precise mechanism of such events is not understood.Here we identify a 12-bp sequence element which is an essential part of the terminator. This 12-bp element contains a shorter 7-bp sequence (the T3 box) which has been conserved between different frog species (16) and which is also present at site T2 (10). This T3 box was required to specify the formation of correct 3' ends. We found that, when inserted between a promoter and site T2, the 12-bp element (but not the T3 box alone) severely reduced the amount of RNA detectable at sites downstream from itself, both in steady-state and pulse-label experiments. In addition, our results indicate that, if taken out of its normal location, the region containing T3 did not appear to constitute a fully efficient terminator since it allowed a significant amount of readthrough transcripts to accumulate. MATERIALS AND METHODSPlasmid construction and mutagenesis. The test vector (Fig. 2) The clustered base changes in the T3 region were created by oligonucleotide-directed mutagenesis essentially as described by Norris et al. (17). Mutant oligonucleotides extending from the BamHI site beyond the T3 box were used to prime the synthesis of a double-stranded DNA cont...

Section: Resultssupporting

confidence: 92%

A 12-base-pair sequence is an essential element of the ribosomal gene terminator in Xenopus laevis.

Labhart¹,

Reeder²

1987

“…Our results from the direct cis stimulation assay (Fig. 3) showing that enhancement by the 60/81-bp repeats is very position dependent refute prior assertions (26,27,42,44) that the 60/81-bp repeats augment transcription equally when located far upstream of the initiation site or when downstream of the initiation site within the transcribed region. These earlier conclusions derived from results obtained with trans competition assays, in which the relative amount of transcription 50-Sg/ml concentration of each of the indicated pairs of plasmids, and transcription was assessed by Si mapping, using the A probe (which hybridizes specifically to transcripts of the A-gene family) and the B probe (which hybridizes specifically to transcripts of the B-gene family).…”

supporting

confidence: 85%

“…The widely accepted view is that the 60/81-bp elements also stimulate transcription from an rDNA promoter in cis and that this stimulation is orientation-and position-independent enhancement (42,44), occurring equally efficiently when the repeats are kilobase pairs distant from the promoter and when they are downstream of the promoter within the transcribed portion of the template (26,27,44,47). These conclusions are based almost entirely on oocyte coinjection experiments using two competing, promoter-bearing templates (13,26,27,45,47); when one also carries the 60/81-bp repeats, the transcriptional balance is shifted toward that promoter and away from the one lacking the repeats (such an assay is illustrated in Fig. 4A, lanes 1 to 3 with two marked rRNA genes).…”

mentioning

confidence: 99%

The Xenopus ribosomal DNA 60- and 81-base-pair repeats are position-dependent enhancers that function at the establishment of the preinitiation complex: analysis in vivo and in an enhancer-responsive in vitro system.

Pape¹,

Windle²,

Mougey³

et al. 1989

Although it is generally believed that the 60-and 81-base-pair (60/81-bp) repeats of the Xenopus laevis ribosomal DNA (rDNA) spacer are position-independent transcriptional enhancers, this has not been shown directly. We have now developed a critical assay which proves that the 60/81-bp repeats do, in fact, stimulate transcription from promoters in cis and that they function in both orientations and when up to 1 kilobase pair from the initiation site. However, contrary to the widely accepted view, these elements are found to be highly position dependent, for they have no net effect when downstream of the initiation site within the transcribed region and they behave as transcriptional silencers of promoters in cis when moved >2 kilobase pairs upstream of the initiation site. The 60/81-bp elements therefore are position-dependent 5' enhancers. We also found that this rDNA enhancer was polymerase I specific and that it was composed of duplicated, individually functional elements. Finally, we report an in vitro system that reproduces both cis enhancement and trans competition by the 60/81-bp repeats. Sequential-addition studies in this system demonstrated that the rDNA enhancer functions in trans at or before establishment of the stable transcription complex, not subsequently at each round of transcription.DNA sequences that augment transcription independent of orientation and at various locations relative to a colinear promoter are termed enhancers. First defined in simian virus 40 (SV40) viral DNA, enhancers have since been identified for a large number of cellular and viral genes transcribed by RNA polymerase II (reviewed in references 18, 25, 40, and 50) and more recently also for a gene transcribed by RNA polymerase III (2). Eucaryotic polymerase II enhancers function 5' and usually also 3' of the initiation site and generally over many kilobase pairs (kb), although their effects can decrease significantly at increasing distance (2,18,25,38,40,41,43,50,60); analogous upstream activation sequences in yeast cells are located within -1 kb upstream of the initiation site and do not appear to function 3' of the initiation site (19,56). Polymerase II enhancers are normally assessed by comparing the level of transcription obtained from two otherwise identical templates, one bearing and one lacking these elements, when introduced separately into cells ( a cis stimulation assay). It has been reported that polymerase II enhancers do not markedly depress transcription of promoters on separate molecules (a trans competition assay [6,49,63,64]); however, some polymerase II enhancers and promoters are now known to share common factors (14,29,35,39). Sequences containing or related to polymerase II enhancer elements have also been shown to act as silencers of cis-located promoters. Although a great deal has been learned about sequences that direct RNA polymerase II enhancement and about factors that bind to these sequences (21, 69; reviewed in references 23, 48, 51, and 68), their mechanisms of action are not yet understood.S...

“…It is useful to compare the enhancer described above with the 60-to 81-bp repeats that have enhancer activity for rRNA transcription in Xenopus oocytes and which also work from upstream of two genes or between two genes (22,23). The two enhancers differ in several respects.…”

Section: Discussionmentioning

confidence: 99%

“…Within them are found a variety of sequence elements that influence the level of transcription. In Xenopus laevis, for example, these include several copies of a sequence closely related to the promoter, dubbed spacer promoters (6,7,22,23,29,33), each followed by about 10 copies of a 60-to 81-base-pair (bp) repeat, that behave in some respects like enhancer elements (22,23,29). The spacer promoters are curious because although they can give rise to transcripts and can stimulate transcription from an adjacent real promoter (6,7,29), the transcription from the spacer promoter seems not to be involved in this stimulation (7,25).…”

mentioning

confidence: 99%

Unusual enhancer function in yeast rRNA transcription.

Johnson

Warner

1989

The rRNA genes in most eucaryotic organisms are present in a tandem array. There is substantial evidence that transcription of one of these genes may not be independent of transcription of others. In particular, in the yeast Saccharomyces cerevisiae, the enhancer of rRNA transcription that lies 2.2 kilobases 5' of the transcription initiation site is at least partly within the upstream transcription unit. The transcription by RNA polymerase II of a wide variety of genes is profoundly influenced by cis-acting short-nucleotide sequences called enhancer elements that can lie upstream or downstream of the transcription unit, at a distance of up to several kilobases. In many cases, the enhancer elements bind protein factors that are necessary for enhancer function. Although a number of models have been proposed (reviewed in reference 30), a clear understanding of enhancer function is not at hand.Two features distinguish rRNA genes from other genes in the eucaryotic cell. They are transcribed by a specific enzyme, RNA polymerase I, and they are arranged in a tandem array. This unique genetic structure has led to speculation that transcription of an individual gene is not independent of transcription of the neighboring genes; e.g., termination at one transcription unit may lead directly to initiation at the neighboring downstream promoter (7,12,15, 26).We have identified a sequence element, specific for RNA polymerase I, that has many of the characteristics of an RNA polymerase II enhancer (9, 10). In this paper, we explore aspects of the function of RNA polymerase I and its enhancer to ask about the parallels between polymerase I and polymerase II enhancers and about the importance of the tandemness of rRNA genes to polymerase I activity.Transcription by RNA polymerase I has been reviewed recently (38). The spacers between individual rRNA transcription units vary in length from 2 kilobase pairs (kb) (yeasts) to >30 kb (mammals). Within them are found a variety of sequence elements that influence the level of transcription. In Xenopus laevis, for example, these include several copies of a sequence closely related to the promoter, dubbed spacer promoters (6,7,22,23,29,33), each followed by about 10 copies of a 60-to 81-base-pair (bp) repeat, that behave in some respects like enhancer elements (22,23,29). The spacer promoters are curious because although they can give rise to transcripts and can stimulate transcription from an adjacent real promoter (6,7,29), the transcription from the spacer promoter seems not to be involved in this stimulation (7,25). Repetitive elements, some related to the promoter, have also been found in the nontranscribed spacer * Corresponding author. t Present address: