Terminator-distal sequences determine the in vitro efficiency of the early terminators of bacteriophages T3 and T7.
Bacteriophages T3 and T7 contain homologous terminators for Escherichia coli RNA polymerase that restrict early phage transcription to the leftmost 20% of the linear phage genomes. These two terminators serve equally well as p-independent terminators in vivo, but their in vitro efficiencies and sensitivity to salt and nucleotide concentrations differ dramatically. Sequence analysis shows that the T7 and T3 terminators differ at only two sites in the region normally accepted as defining terminator function. In order to determine which structural features of these two terminators are responsible for their functional differences, a series of hybrid terminators were constructed in which structural features of the two terminators were systematically interchanged. Transcription of hybrid terminator templates revealed that sequences downstream of the termination release sites are responsible for the differences in efficiency of in vitro termination. These sequences also determine the sensitivity of these terminators to elevated salt concentrations and to alterations of substrate concentrations. Alteration of the sequences in the region between three and seven nucleotides downstream of the final T7Te release site is sufficient to reduce termination efficiency to that of T3Te, and point mutations in this region yield terminators with intermediate efficiency. Hence, the determinants of p-independent terminator efficiency in vitro must include elements of the transcription complex other than the structure of the 3' end of the transcript. The termination differences between T7Te, T3Te, and their hybrid derivatives are overcome in vivo; all of these sites become very efficient. This finding further supports the hypothesis that protein factors or other cellular features enhance the efficiency and specificity of p-independent terminators in vivo.
Telesnitsky
A
A
Department of Biochemistry, University of California, Berkeley 94720.
Chamberlin
M J
MJ
D004247:DNA / Q000032:analysis; D012321:DNA-Directed RNA Polymerases / Q000235:genetics; D003851:Deoxyribonucleases; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005809:Genes, Regulator; D006868:Hydrolysis; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization; D010446:Peptide Fragments / Q000032:analysis; D012333:RNA, Messenger / Q000032:analysis; D012689:Sequence Homology, Nucleic Acid; D013604:T-Phages / Q000235:genetics*; D013698:Templates, Genetic; D013728:Terminator Regions, Genetic; D014158:Transcription, Genetic
D010446:Peptide Fragments; D012333:RNA, Messenger; D004247:DNA; D012321:DNA-Directed RNA Polymerases; D003851:Deoxyribonucleases