A novel LBP-1-mediated restriction of HIV-1 transcription at the level of elongation in vitro.
The cellular factor, LBP-1, can repress HIV-1 transcription by preventing the binding of TFIID to the promoter. Here we have analyzed the effect of recombinant LBP-1 on HIV-1 transcription in vitro by using a "pulse-chase" assay. LBP-1 had no effect on initiation from a preformed preinitiation complex and elongation to position +13 ("pulse"). However, addition of LBP-1 after RNA polymerase was stalled at +13 strongly inhibited further elongation ("chase") by reducing RNA polymerase processivity. Severe mutations of the high affinity LBP-1 binding sites between -4 and +21 did not relieve the LBP-1-dependent block. However, LBP-1 could bind independently to upstream low affinity sites (-80 to -4), suggesting that these sites mediate the effect of LBP-1 on elongation. These results demonstrate a novel function of LBP-1, restricting HIV-1 transcription at the level of elongation. In addition, Tat was found to suppress the antiprocessivity effect of LBP-1 on HIV-1 transcription in nuclear extracts. These findings strongly suggest that LBP-1 may provide a natural mechanism for restricting the elongation of HIV-1 transcripts and that this may be a target for the action of Tat in enhancing transcription.
Parada
C A
CA
Laboratory of Biochemistry and Molecular Biology, Rockefeller University, New York, New York 10021.
Yoon
J B
JB
Roeder
R G
RG
D001483:Base Sequence; D004268:DNA-Binding Proteins / Q000502:physiology*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D015967:Gene Expression Regulation, Viral; D015696:Gene Products, tat / Q000378:metabolism; D015497:HIV-1 / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D012367:RNA, Viral / Q000096:biosynthesis*; D011994:Recombinant Proteins; D012097:Repressor Proteins / Q000502:physiology*; D013329:Structure-Activity Relationship; D016385:TATA Box; D014157:Transcription Factors; D014158:Transcription, Genetic; D054322:tat Gene Products, Human Immunodeficiency Virus
D004268:DNA-Binding Proteins; D015696:Gene Products, tat; D012367:RNA, Viral; D011994:Recombinant Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; C085968:UBP1 protein, human; D054322:tat Gene Products, Human Immunodeficiency Virus; D012321:DNA-Directed RNA Polymerases