Tripartite sequences within and 3' to the sea urchin H2A histone gene display properties associated with a transcriptional termination process.

Abstract

We have defined a DNA sequence that behaves as an RNA polymerase II termination signal by using the human HeLa cell transient expression system. Surprisingly, this sequence is tripartite, including part of the coding region of the sea urchin H2A histone gene together with two separate sequences in the 3' flanking region of the gene. We demonstrate that this signal functions both in its normal gene environment and also when placed within the human a-globin gene. However, we have failed to detect a discrete 3' terminus. Rather, our data indicate the, presence of an extremely heterogeneous series of nonpolyadenylated RNAs. These heterogeneous nonpolyadenylated RNAs are stable when transcribed from the intact histone gene but are highly unstable within the human a-globin gene. This provides evidence for the role of poly(A) in the stability of mnRNA.Initiation of transcription in eucaryotic RNA polymerase II (polII) genes is a highly regulated and in some instances well-defined process (see reference 28 for a review). In contrast, termination of transcription in polII genes is ill defined and may not be a regulated process. The principal cause of this uncertainty is the rapid posttranscriptional processing of primary RNA transcripts to mature mRNA (see reference 40 for a review). The fact that introns are spliced out of the polII gene transcript is well documented. Similarly, the generation of mRNA 3' termini for both polyadenylated [poly(A)+] mRNA and nonpolyadenylated [poly(A)-] histone mRNA by a processing event has been recently established (see references 5 and 44 for reviews).The best evidence for processing of mRNA 3' ends comes from studies on histone mRNA. Thus, the existence of a histone mRNA 3' processing activity in Xenopus laevis oocytes and Drosophila melanogaster tissue culture cell extracts has been clearly demonstrated (4,25,43). Furthermore, it has been shown that this activity is associated with a small nuclear RNA U7 (12, 52) in a similar way to the involvement of the small nuclear RNA Ul in intronic splicing (24,27,42,47). These results indicate that transcriptional termination is likely to occur separately from 3'-end processing in histone genes. Evidence of a similar nature in poly(A)+ mRNA genes is now becoming available. Moore and Sharp (37) have recently shown that synthetic RNAs extending beyond an adenovirus poly(A) site can be efficiently and accurately cleaved and polyadenylated by using in vitro cell extracts.Further evidence for transcriptional termination separate from nmRNA 3'-end formation comes from hybridization analysis of pulse-labeled RNA transcripts. Using these techniques, Nevins and Darnell (38) and Fraser et al. (11) deduced that the major late gene transcripts of adenovirus extend beyond the various late mRNA poly(A) sites to near the end of the viral genome. Similarly, transcription extends beyond the adenovirus early region 2 and 4 poly(A) sites and the simian virus 40 (SV40) late poly(A) site (10, 39). These viral transcription experiments were performed on pulse-* Corr...

Oligonucleotide sequence signaling transcriptional termination of vaccinia virus early genes.

“…In fact, there is preliminary evidence that ATTTTTAT is part of a longer RNA polymerase II termination signal in yeast (4) and in mammals (6) and may regulate termination in an in vitro system derived from HeLa cells (25). Thymidine-rich clusters also form part of a putative tripartite sequence proposed for sea urchin histone mRNA termination (5).…”

“…The available information suggests that termination is heterogeneous and may occur far downstream of the mature, processed 3' end of the transcript (1-3). Several putative termination sequences have been proposed (4)(5)(6)(7)(8), but in general processing and termination mechanisms have not been adequately distinguished because of difficulties in developing an in vitro system. However, termination was shown to occur in an in vitro system derived from vaccinia virus particles (9).…”

An intact histone 3'-processing site is required for transcription termination in a mouse histone H2a gene.

“…They suggested that termination of transcription in the sea urchin H2a gene injected into Xenopus oocytes occurred about 200 nt downstream of the gene. This same sequence has been shown to terminate transcription of heterologous genes in mammalian cells in the absence of a 3'-processing signal (8,25), suggesting that the mechanism of transcription termination may differ between histone genes and genes encoding polyadenylated mRNAs.…”

Transcription termination at the chicken beta H-globin gene.

“…RNA polymerase II, like RNA polymerase III, terminates at dT clusters without the assistance of factors (7, 11). The mouse fm'j-globin, human a2-globin, and sea urchin H2A histone genes all direct termination by utilizing signals upstream of multiple transcription endpoints reminiscent of p-dependent terminators in E. coli (23,34,59). Even the alternate stem-loop motif of procaryotic attenuators appears to be used in a functionally analogous way by RNA polymerase II in regulation of "premature" termination in simian virus 40 (19) and possibly also in c-myc (13).…”

“…However, one generalization emerges if attention is restricted to the 3' ends of the transcription units (i.e., eliminating attenuation from consideration). Transcription does not terminate at a specific site but declines gradually to zero, reflecting multiple sites of polymerase release (2,6,14,18,22,23,32,44,47,55,58,60). This suggests that the termination signal in these genes specifies loss of transcriptional processivity rather than termination per se (44).…”