TAT FUNCTIONS TO STIMULATE THE ELONGATION PROPERTIES OF TRANSCRIPTIONCOMPLEXES PAUSED BY THE DUPLICATED TAR RNA ELEMENT OF HUMAN-IMMUNODEFICIENCY-VIRUS-2
Lf. Garciamartinez et al., TAT FUNCTIONS TO STIMULATE THE ELONGATION PROPERTIES OF TRANSCRIPTIONCOMPLEXES PAUSED BY THE DUPLICATED TAR RNA ELEMENT OF HUMAN-IMMUNODEFICIENCY-VIRUS-2, Journal of Molecular Biology, 254(3), 1995, pp. 350-363
In this study we have defined the in vitro requirements for transcript
ional regulation of the HIV-2 LTR in response to the HIV-1 and HIV-2 T
ar proteins and addressed potential mechanisms of Tat function. HIV-2
contains a duplicated TAR RNA stem-loop structure in contrast to the s
ingle stem-loop structure found in HIV-1 TAR RNA. We demonstrated that
the HIV-2 proximal TAR RNA stem-loop structure was more important for
in vitro transcriptional activation by the HIV-1 and HIV-2 Tat protei
ns than the distal TAR RNA stem-loop though this downstream TAR elemen
t itself was able to confer Tat-responsiveness. The role of the two HI
V-2 TAR RNA stem-loop bulge sequences was less critical than the loop
sequences for in vitro transcriptional activation by Tat. In addition,
we demonstrated that replacing the HIV-2 TATA element with that of HI
V-1 markedly reduced the overall level of Tat activation. The role of
the Tat-1 and Tat-2 proteins on the synthesis of HIV-1 and HIV-2 promo
ter proximal and promoter distal transcripts was then investigated. In
contrast to the HIV-1 promoter, the HIV-2 promoter generated abundant
levels of short transcripts in in vitro transcription assays likely d
ue to the structure of its duplicated TAR element. Both Tat-1 and Tat-
2 increased the level of transcripts extending to the end of the HIV-1
and HIV-2 TAR elements as well as the level of transcripts extending
more than 500 nucleotides from the transcription initiation site. Howe
ver, the synthesis of transcripts within 30 nucleotides of the HIV-2 L
TR transcription initiation site was unchanged in either the presence
or absence of Tat while the level of transcripts extending increasing
distances from the HIV-2 LTR transcription initiation site were progre
ssively stimulated in the presence of Tat. Though the HIV-1 Tat protei
n was a stronger inducer of HIV-1 LTR transcription than the HIV-2 Tat
protein, we did not detect differences in the binding of these protei
ns to the HIV-1 and HIV-2 TAR RNAs. This suggested that differences in
their transactivation properties may be due to alterations in their a
ssociation with RNA polymerase II or associated elongation factors. Th
ese results indicate that the effects of both Tat-1 and Tat-2 on the H
IV-2 LTR result in enhanced promoter clearance of RNA polymerase II tr
anscription complexes which are paused by the HIV-2 TAR element and th
at the TAR element is possibly the site for loading of Tat onto the tr
anscriptional elongation complex. (C) 1995 Academic Press Limited