Poly(A)-Driven and Poly(A)-Assisted Termination: Two Different Modes of Poly(A)-Dependent Transcription Termination

Our understanding of transcription termination by eukaryotic RNA polymerase II is steadily increasing as more terminators from protein-coding genes are characterized. One theme that has emerged from the characterization of several transcription units is the requirement of a functional poly(A) signal for efficient termination to proceed (11, 32, 36, 66). In many cases, if any part of the poly(A) signal is damaged by mutation, transcription termination is impaired (11, 15, 36, 66). In other cases, however, a correlation between the efficiency of polyadenylation and the efficiency of termination is not apparent (7, 17, 61). One interpretation of poly(A)-dependent termination is that some aspect of the 3′-end processing reaction is involved in potentiating termination by the polymerase. Possibly the 3′-end cleavage event activates a termination factor (11, 51). Alternatively, assembly of the cleavage-polyadenylation complex at the polymerase surface may signal termination (41). The nature of the actual termination event potentiated by the poly(A) signal is unclear. Logan et al. (36) have suggested that interaction with the poly(A) signal returns the transcriptional apparatus to a prior state of low processivity. Thus altered, the polymerase dissociates stochastically from the template. This model is based on the observation that successful transcription of eukaryotic pre-mRNA-coding genes requires not only the initiation of transcription at the promoter but also the establishment of a processive elongation complex. The commencement of elongation without the establishment of processivity results in premature termination of transcription within the first several hundred base pairs downstream of the promoter (8, 28, 33, 40, 42, 47–49, 52, 53, 67, 69). The model of Logan et al. (36) is consistent with the behavior of transcription units in which transcription terminates heterogeneously, beginning immediately downstream of the poly(A) site (1, 22). However, no candidate Logan-type termination region has yet been definitively characterized. On the other hand, not explained by the model of Logan et al. (36) are the many instances in which transcription proceeds for great distances past the poly(A) site with little decrease in processivity (12, 27, 39, 44). In the well-characterized case of a chimeric plasmid containing the simian virus 40 (SV40) early transcription unit (10, 11), little termination occurs beyond the poly(A) site unless a special termination element is encountered. However, a functional poly(A) signal is required in order for the downstream termination element to be fully effective (10, 11). Thus, efficient termination in this case is potentiated by a functional poly(A) signal but is realized only upon encountering the additional downstream terminator. Downstream terminator elements may not be uncommon and apparently take a variety of forms. The one described by Connelly and Manley (10, 12) is the protein binding site, CCAAT, probably bound by CP1. Ashfield et al. (2, 3) have also characterized a protein binding site (for MAZ) that acts as a downstream terminator. Additional downstream terminators that probably act at the level of DNA sequence per se rather than as protein binding sites have previously been described (16, 60). How these elements act is unknown, but their collaboration with poly(A) sites has led to the suggestion that they are pause sites, whose role is to delay the polymerase until polyadenylation has had sufficient time to occur (11, 51). Indeed, independent evidence suggests that one of these elements is in fact a pause site (16). Although the downstream elements that have been characterized so far depend on functional upstream poly(A) signals for full efficiency, in at least one case, the CCAAT box, the signal is partially effective on its own. Moreover, duplicating the element increases its effectiveness when no poly(A) site is present (12). Thus, as has previously been suggested (14, 55), it is possible that some terminators of processive RNA polymerase II transcription do not require the assistance of a poly(A) signal. In the present work, we characterized extensively the elements responsible for termination of chicken βH-globin gene transcription. We found that termination of transcription for this gene is best described by the model of Logan et al. (36) and thus differs from that of most genes so far characterized. Termination was poly(A) dependent, and no additional downstream termination element was required. Moreover, termination appeared to commence promptly after the poly(A) site; no significant span of processive transcription intervened between the poly(A) signal and the region of termination. In comparison to the previous work described above, these results suggest that the following two modes of transcription termination exist:poly(A)-driven, terminator-independent termination and poly(A)-assisted, terminator-dependent termination. We therefore carried out a functional comparison of the 3′-flanking regions of the βH-globin and SV40 early transcription units. The comparison confirmed the inferred difference between the two termination modes and showed moreover that the poly(A)-driven mode of the βH-globin gene depends on the presence of an upstream termination enhancer which is not present in the SV40 early region.