Phosphorylation of the RNA polymerase II C-terminal domain (CTD) by cyclin-dependent kinases is important for productive transcription. Here we determine the crystal structure of Cdk12/CycK and analyse its requirements for substrate recognition. Active Cdk12/CycK is arranged in an open conformation similar to that of Cdk9/CycT but different from those of cell cycle kinases. Cdk12 contains a C-terminal extension that folds onto the N- and C-terminal lobes thereby contacting the ATP ribose. The interaction is mediated by an HE motif followed by a polybasic cluster that is conserved in transcriptional CDKs. Cdk12/CycK showed the highest activity on a CTD substrate prephosphorylated at position Ser7, whereas the common Lys7 substitution was not recognized. Flavopiridol is most potent towards Cdk12 but was still 10-fold more potent towards Cdk9. T-loop phosphorylation of Cdk12 required coexpression with a Cdk-activating kinase. These results suggest the regulation of Pol II elongation by a relay of transcriptionally active CTD kinases.

The replication of many retroviruses is mediated by a transcriptional activator protein, Tat, which activates RNA polymerase II at the level of transcription elongation. Tat interacts with Cyclin T1 of the positive transcription-elongation factor P-TEFb to recruit the transactivation-response TAR RNA, which acts as a promoter element in the transcribed 5' end of the viral long terminal repeat. Here we present the structure of the cyclin box domain of Cyclin T1 in complex with the Tat protein from the equine infectious anemia virus and its corresponding TAR RNA. The basic RNA-recognition motif of Tat adopts a helical structure whose flanking regions interact with a cyclin T-specific loop in the first cyclin box repeat. Together, both proteins coordinate the stem-loop structure of TAR. Our findings show that Tat binds to a surface on Cyclin T1 similar to where recognition motifs from substrate and inhibitor peptides were previously found to interact within Cdk-cyclin pairs.

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Reverse transcriptases (RTs) ␣␤ and ␤ from avian Rous sarcoma virus (RSV) harbor an integrase domain which is absent in nonavian retroviral RTs. RSV integrase contains a nuclear localization signal which enables the enzyme to enter the nucleus of the cell in order to perform integration of the proviral DNA into the host genome. In the present study we analyzed the subcellular localization of RSV RT, since previous results indicated that RSV finishes synthesis of the proviral DNA in the nucleus. Our results demonstrate that the heterodimeric RSV RT ␣␤ and the ␤ subunit, when expressed independently, can be detected in the nucleus, whereas the separate ␣ subunit lacking the integrase domain is prevalent in the cytoplasm. These data suggest an involvement of RSV RT in the transport of the preintegration complex into the nucleus. In addition, to analyze whether the integrase domain, located at the carboxyl terminus of ␤, exhibits integration activities, we investigated the nicking and joining activities of heterodimeric RSV RT ␣␤ with an oligodeoxynucleotide-based assay system and with a donor substrate containing the supF gene flanked by the viral long terminal repeats. Our data show that RSV RT ␣␤ is able to perform the integration reaction in vitro; however, it does so with an estimated 30-fold lower efficiency than the free RSV integrase, indicating that RSV RT is not involved in integration in vivo. Integration with RSV RT ␣␤ could be stimulated in the presence of human immunodeficiency virus type 1 nucleocapsid protein or HMG-I(Y).After a retrovirus infects a cell, retroviral core particles are released into the cytoplasm, where the viral RNA is reverse transcribed into double-stranded DNA within a nucleoprotein structure, designated the preintegration complex (PIC) (3,4,7,43,44,60). In addition to the viral DNA and integrase (IN), PICs include several other viral and cellular proteins, among them the viral enzyme reverse transcriptase (RT), the viral nucleocapsid (NC) protein, and the cellular nonhistone DNAbinding protein HMG-I(Y) (8,16,17,23). For retroviral integration to occur, the newly synthesized viral DNA must associate with the host genome. Human immunodeficiency virus (HIV) is not dependent on host cell division for integration and virus propagation but enters the nucleus via transport through the nuclear pore (46). The viral IN and the Vpr protein carry nuclear localization signals (NLSs) that are thought to be responsible for transport of the HIV PIC into the nucleus (10,20,22,41,56,63). A contribution of the matrix protein (MA) has also been suggested (23,26,49).In contrast to HIV, oncogenic retroviruses such as murine leukemia virus (MLV) and the avian sarcoma and leukosis virus (ASLV) are dependent on target cell proliferation for productive replication (27,35,37,61). For MLV it was demonstrated that integration of viral DNA requires passage through mitosis, indicating the necessity of nuclear envelope breakdown (47, 57). For ASLV it was suggested that the viral DNA can be synthesized and integ...

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