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

ABSTRACT

We mapped the elements that mediate termination of transcription downstream of the chicken β^H- and β^A-globin gene poly(A) sites. We found no unique element and no segment of 3′-flanking DNA to be significantly more effective than any other. When we replaced the native 3′-flanking DNA with bacterial DNA, it too supported transcription termination. 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. We also studied premature termination by poorly processive polymerases close to the promoter. The rate of premature termination varied for different DNA sequences. 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 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. In the SV40 early transcription unit, essentially no termination occurs downstream of the poly(A) site unless a special termination element is present. The difference between the β^H-globin and SV40 modes of termination is governed by sequences in the upstream DNA. For maximum efficiency, the β^H-globin poly(A) signal required the assistance of upstream enhancing sequences. 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. 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.

ACKNOWLEDGMENTS

We thank E. Erickson for carrying out some preliminary work at the inception of this study, N. Nguyen for constructing some of the plasmids used and for communicating unpublished results, and R. Landick for providing plasmid pRL542.

This work was supported by NIH grant GM50863.