In vitro activity of the baculovirus late expression factor LEF-5.
The baculovirus late expression factor LEF-5 has a zinc ribbon that is homologous to a domain in the eukaryotic transcription elongation factor SII. To determine whether LEF-5 is an elongation factor, we purified it from a bacterial overexpression system and added it to purified baculovirus RNA polymerase. LEF-5 increased transcription from both late and very late viral promoters. Two acidic residues within the zinc ribbon were essential for stimulation. Unlike SII, however, LEF-5 did not appear to enable RNA polymerase to escape from intrinsic pause sites. Furthermore, LEF-5 did not increase transcription in the presence of small DNA-binding ligands that inhibit elongation in other systems or viral DNA-binding proteins which inhibit the baculovirus RNA polymerase. Exonuclease activity assays revealed that baculovirus RNA polymerase has an intrinsic exonuclease activity, but this was not increased by the addition of LEF-5. Initiation assays and elongation assays using heparin to prevent reinitiation indicated that LEF-5 was active only in the absence of heparin. Taken together, these results suggest that LEF-5 functions as an initiation factor and not as an elongation factor.
Guarino
Linda A
LA
Departments of Biochemistry, Texas A&M University, 2128 TAMU, College Station, TX 77843-2128, USA. lguarino@tamu.edu
Dong
Wen
W
Jin
Jianping
J
D016367:Baculoviridae / Q000235:genetics*; D004247:DNA / Q000378:metabolism; D006493:Heparin / Q000494:pharmacology; D010445:Peptide Elongation Factors / Q000502:physiology*; D010448:Peptide Initiation Factors / Q000502:physiology*; D012319:RNA Polymerase II / Q000502:physiology; D014158:Transcription, Genetic; D014764:Viral Proteins / Q000302:isolation & purification / Q000502:physiology*
D010445:Peptide Elongation Factors; D010448:Peptide Initiation Factors; D014764:Viral Proteins; D006493:Heparin; D004247:DNA; D012319:RNA Polymerase II