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. ( 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). On the other hand, not explained by the model of Logan et al.
However, no candidate Logan-type termination region has yet been definitively characterized. ( 36) is consistent with the behavior of transcription units in which transcription terminates heterogeneously, beginning immediately downstream of the poly(A) site ( 1, 22). 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). 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. Thus altered, the polymerase dissociates stochastically from the template. ( 36) have suggested that interaction with the poly(A) signal returns the transcriptional apparatus to a prior state of low processivity. The nature of the actual termination event potentiated by the poly(A) signal is unclear. Alternatively, assembly of the cleavage-polyadenylation complex at the polymerase surface may signal termination ( 41). Possibly the 3′-end cleavage event activates a termination factor ( 11, 51). 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. In other cases, however, a correlation between the efficiency of polyadenylation and the efficiency of termination is not apparent ( 7, 17, 61). In many cases, if any part of the poly(A) signal is damaged by mutation, transcription termination is impaired ( 11, 15, 36, 66). 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). Our understanding of transcription termination by eukaryotic RNA polymerase II is steadily increasing as more terminators from protein-coding genes are characterized. These studies were facilitated by a rapid, improved method of run-on transcription analysis, based on the use of a vector containing two G-free cassettes. Moreover, the SV40 early poly(A) signal also drove termination in β H-globin style when it was placed in a β H-globin sequence context. For maximum efficiency, the β H-globin poly(A) signal required the assistance of upstream enhancing sequences. The difference between the β H-globin and SV40 modes of termination is governed by sequences in the upstream DNA. In the SV40 early transcription unit, essentially no termination occurs downstream of the poly(A) site unless a special termination element is present. The poly(A)-driven termination described here differs dramatically from the poly(A)-assisted termination previously described for the simian virus 40 (SV40) early transcription unit. However, the efficiencies of poly(A)-driven termination and promoter-proximal premature termination varied similarly on different DNAs, suggesting that poly(A)-driven termination functions by returning the transcription complex to a form which resembles a prior state of low processivity. The rate of premature termination varied for different DNA sequences.
We also studied premature termination by poorly processive polymerases close to the promoter. Termination in the bacterial DNA depended on a functional poly(A) signal, which apparently compelled termination to occur in the downstream DNA with little regard for its sequence. When we replaced the native 3′-flanking DNA with bacterial DNA, it too supported transcription termination. We found no unique element and no segment of 3′-flanking DNA to be significantly more effective than any other. We mapped the elements that mediate termination of transcription downstream of the chicken β H- and β A-globin gene poly(A) sites.
0 kommentar(er)
