Proc. Natl. Acad. Sci. USA Vol. 92, pp. 2408-2412, March 1995 Biochemistry The RNA element encoded by the trans-activation-responsive region of human immunodeficiency virus type 1 is functional when displaced downstream of the start of transcription (RNA-protein interaction/trans-activation/Tat protein/transcription elongation) MARK J. CHURCHER, ANTHONY D. LOWE, MICHAEL J. GAIT, AND JONATHAN KARN* Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 20H, United Kingdom Communicated by Sydney Brenner, University of Cambridge School of Clinical Medicine, Cambridge, United Kingdom, December 7, 1994 ABSTRACT The human immunodeficiency virus type 1 a TAR RNA element placed anywhere downstream of the (HIV-1) trans-activator protein, Tat, specifically stimulates promoter. To examine whether TAR is functional at a dis- transcription from the viral long terminal repeat. Tat binds to tance, we prepared templates carrying duplicated TAR ele- an RNA stem-loop structure encoded by the trans-activation ments. The contributions ofvarious control elements encoded response region (TAR). To test whether TAR is functional by TAR were then measured by introducing mutations into when displaced downstream of the start of transcription, we either the upstream or the downstream TAR elements. The assayed a series of templates carrying duplicated TAR ele- results demonstrate that TAR RNA elements can allow effi- ments in cell-free transcription systems. When the normally cient recruitment of Tat and the activation of transcriptional positioned TAR element (TAR-1) is inactivated by mutations elongation even when they are placed several hundred nucle- in either the Tat binding site or the apical loop sequence, otides downstream of the transcription start site. which acts as the binding site for a cellular factor, trans- activation can be rescued by a wild-type TAR element placed downstream (TAR-2). The TAR-2 element is functional even MATERIALS AND METHODS when placed >200 nt downstream of TAR-1. TAR comple- Template DNAs. Test plasmids were derived from the mentation experiments have also shown that a functional TAR pMAG-10 plasmid (5), which carries a synthetic terminator element requires both an intact Tat binding site and an intact sequence (T) located downstream of the viral LTR (Fig. 1). apical loop sequence. For example, if TAR-1 carries a muta- Duplicated TAR elements were inserted between the HindIII tion in the loop element it cannot be rescued by a TAR-2 and Nar I sites- downstream of the viral LTR. Sequences element carrying a mutation in the Tat binding site. Substi- derived from the bacterial chloramphenicol acetyltransferase tution mutations in TAR-1 show that the 5' half of TAR also (CAT) gene were used to replace sequences in TAR in order encodes an essential DNA element which is required for to map critical DNA elements and to provide "spacers" to efficient transcription initiation. These results strongly sug- move TAR-2 to different positions. gest that Tat and cellular cofactors which bind TAR RNA Cell-Free Transcription. Transcription reactions (50 ,ul) associate with the transcription complex during its transit were performed essentially as described (5). Templates were through TAR. linearized by cleavage with Xba I. After preincubation for 15 or 20 min, the reaction mixtures were incubated for 30 min in The human immunodeficiency virus type 1 (HIV-1) Tat protein the presence of [a-32P]UTP. Transcripts were fractionated by provides the first example of a viral protein that regulates electrophoresis in 6% polyacrylamide gels containing 7 M transcriptional elongation in eukaryotic cells. In the absence of urea, 90 mM Tris base, 89 mM boric acid, and 2 mM EDTA Tat, the majority of the transcriptional complexes formed at the (pH 8.3). The gels were dried and then exposed to x-ray film HIV promoter stall or disengage near the start of transcription, for the times indicated in the figure legends. whereas in the presence of Tat there is a dramatic increase in the production of full-length viral transcripts (1-7). Deletion analysis of the viral long terminal repeat (LTR) RESULTS showed that Tat activity requires the trans-activation-respon- Phenotypes ofTAR Mutants. Detailed analysis of the role of sive region (TAR) (8-12). TAR encodes an RNA stem-loop TAR in the trans-activation mechanism is now possible be- structure which acts as a binding site for the Tat protein (13). cause of the development of efficient cell-free transcription Extensive mutagenesis studies have now defined the key systems that respond to Tat (4-7, 20). In an earlier paper (5) elements required for TAR recognition and demonstrated we used a cell-free system derived from HeLa cells to dem- that there is a direct correlation between Tat binding to TAR onstrate that the Tat-stimulated RNA polymerase can read RNA and trans-activation (reviewed in ref. 14). In addition to through a synthetic terminator sequence (T) placed down- acting as a binding site for Tat protein, the apical portion of stream of the start of transcription. The presence of T caused TAR RNA also acts as a binding site for cellular RNA-binding "50% of the transcribing polymerases to disengage prema- proteins that participate in trans-activation (15, 16). DNA turely from the template. Addition of recombinant Tat protein control elements essential for transcription initiation may also to the reaction stimulated the production of run-off product overlap TAR (3, 17, 18). (p) by >30-fold (Fig. 1). One likely mechanism for trans-activation is that Tat forms Tat-dependent trans-activation in vitro has the same se- a modified transcription complex together with RNA poly- quence requirements seen in vivo (5). In the experiments that merase, TAR RNA, and cellular factors (1, 5, 19-21). If this follow, we have inactivated TAR by either the G26-C39 -> C-G model is correct, then it should be possible to introduce Tat via Abbreviations: CAT, chloramphenicol acetyltransferase; HIV, human The publication costs of this article were defrayed in part by page charge immunodeficiency virus; LTR, long terminal repeat; TAR, trans- payment. This article must therefore be hereby marked "advertisement" in activation-responsive region. accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed. 2408 Downloaded by guest on September 24, 2021 Biochemistry: Churcher et al Proc. Natl. Acad. Sci. USA 92 (1995) 2409 LTR TA TAR-1 TAR-2 p LTR \ gag -453 +1 224 666 ----.-w_ -453 + 1 102 222 664 uG G1 u Template Template [ Template C-GA (mLG) MTX MTX MTX I~~~~~~~~~~~~~~~~~ C-G 1 63 64 MTX MTX MTX MTX MTX MTX MTX MTX G-C ITAR WT mGCmL 53 60 61 62 1 55 54 56 A-U TAR-1 WT WT mGC mGC WT WT mLG mLG (mGC) TAR-22 WT mmGC WT mGC [ - mLG WT mLG A-U -622 Tat G-C -1+1-1+1-1+1-1+.:++ .... A-U 527 -404 TAR RNA .- AU -309 A U G-C G-C G-C U-A ..w.=a C-G --217 C-G C- C-G _201 A-U -190 G-C -180 G-C G-C C-GUUUUUUUUU -160 Terminator (X) I FIG. 2. Rescue of mutant TAR elements by a wild-type TAR element downstream of the promoter. (Left) Assay of templates FIG. 1. Cell-free transcription of templates carrying the HIV LTR. placed carrying the mGC mutation in either TAR-1 or TAR-2. Note that (Upper) Structure of template DNAs. Each plasmid contains a syn- MTX-61, which carries the mGC mutation in TAR-1 and a wild-type of the start of thetic terminator (T) inserted downstream transcription. TAR-2, responds strongly to Tat, whereas the control template Templates carried either the mGC (G26.C39 -> C.G) mutation, which MTX-62, in which both TAR-1 and TAR-2 are inactivated by the mGC reduces Tat >14-fold, or the mLG -- UUU at binding by (GGG mutation, is unable to respond to Tat. MTX-53 is a control template 32-34) mutation, which does not affect Tat binding. Transcription carrying wild-type TAR-1 and TAR-2 elements. MTX-60 carries a reactions were performed in the presence of 0 (-) or 200 ng (+) wild-type TAR-1 element and a TAR-2 element with the mGC recombinant Tat protein. p, Runoff transcript; T, transcripts ending at mutation. (Right) Assay of templates carrying the mLG mutation in terminator. Gel was exposed to x-ray film at -70°C for 15 hr. either TAR-1 or TAR-2. The MTX-54 template, which carries the mLG mutation in TAR-1 and a wild-type TAR-2, responds efficiently (mGC) mutation in the Tat binding site or by the GGG to Tat, whereas MTX-56, which carries the mLG mutation in both UUU (mLG) mutation at 32-34 in the apical loop sequence TAR-1 and TAR-2, does not respond significantly to Tat. MTX-1 is (Fig. 1). The mGC mutation reduces Tat binding to TAR RNA a control template carrying a single wild-type TAR element, whereas by >14-fold and inactivates the viral LTR in reporter systems, MTX-55 is a control template carrying a wild-type TAR-1 element and whereas the mLG mutation does not affect Tat binding but an inactive TAR-2 element. -, No Tat protein; +, 200 ng of Tat inactivates trans-activation in vivo (22). protein; p, runoff transcripts; T, transcripts ending at the synthetic terminator. Transcripts were fractionated in a 6% polyacrylamide gel Tat Can Be Introduced via a Displaced TAR RNA Element. and exposed to x-ray film at -70°C for 15 hr. If Tat binds to the TAR RNA which is produced by an elongating RNA polymerase, then it should also be possible to contrast, MTX-62, which carries the mGC mutation in both introduce Tat when TAR is placed considerably downstream TAR-1 and TAR-2, has negligible activity.