The HEXIM1 protein inhibits the kinase activity of P-TEFb (CDK9/cyclin T) to suppress RNA polymerase II transcriptional elongation in a process that specifically requires the 7SK snRNA, which mediates the interaction of HEXIM1 with P-TEFb. In an attempt to define the sequence requirements for HEXIM1 to interact with 7SK and inactivate P-TEFb, we have identified the first 18 amino acids within the previously described nuclear localization signal (NLS) of HEXIM1 as both necessary and sufficient for binding to 7SK in vivo and in vitro. This 7SK-binding motif was essential for HEXIM1's inhibitory action, as the HEXIM1 mutants with this motif replaced with a foreign NLS failed to interact with 7SK and P-TEFb and hence were unable to inactivate P-TEFb. The 7SK-binding motif alone, however, was not sufficient to inhibit P-TEFb. A region C-terminal to this motif was also required for HEXIM1 to associate with P-TEFb and suppress P-TEFb's kinase and transcriptional activities. The 7SK-binding motif in HEXIM1 contains clusters of positively charged residues reminiscent of the arginine-rich RNA-binding motif found in a wide variety of proteins. Part of it is highly homologous to the TAR RNA-binding motif in the human immunodeficiency virus type 1 (HIV-1) Tat protein, which was able to restore the 7SK-binding ability of a HEXIM1 NLS substitution mutant. We propose that a similar RNA-protein recognition mechanism may exist to regulate the formation of both the Tat-TAR-P-TEFb and the HEXIM1-7SK-P-TEFb ternary complexes, which may help convert the inactive HEXIM1/7SK-bound P-TEFb into an active one for Tat-activated and TAR-dependent HIV-1 transcription.During transcription by RNA polymerase II (Pol II), phosphorylation of the carboxy-terminal domain (CTD) of the largest subunit of Pol II by the positive transcriptional elongation factor b (P-TEFb) is crucial for the transition from the abortive to the productive phase of transcriptional elongation, leading to the generation of full-length RNA transcripts (for reviews, see references 7 and 14). Active P-TEFb is composed of CDK9 and its regulatory subunit, cyclin T1 (CycT1) (14). Unlike other CDKs and their cyclin partners whose functions are closely related to the cell cycle regulation, P-TEFb is constitutively expressed and, hence, its protein level varies little throughout the cell cycle (5). Not only is P-TEFb essential for the expression of most protein-encoding genes (2, 16), but also it is indispensable for the replication of human immunodeficiency virus type 1 (HIV-1), since it is a specific host cellular cofactor for the viral Tat protein (7,14). P-TEFb is recruited by Tat to the HIV-1 long terminal repeat (LTR) through the formation of a stable ternary complex consisting of P-TEFb, Tat, and the TAR RNA stem-loop structure located at the 5Ј end of the nascent viral transcript (14). Once recruited, P-TEFb phosphorylates the Pol II CTD and stimulates the production of full-length HIV-1 transcripts.Not every P-TEFb complex in the cell can function as a transcription factor...

Bacteria assemble complex structures by targeting proteins to specific subcellular locations. The protein coat that encases Bacillus subtilis spores is an example of a structure that requires coordinated targeting and assembly of more than 24 polypeptides. The earliest stages of coat assembly require the action of three morphogenetic proteins: SpoIVA, CotE, and SpoVID. In the first steps, a basement layer of SpoIVA forms around the surface of the forespore, guiding the subsequent positioning of a ring of CotE protein about 75 nm from the forespore surface. SpoVID localizes near the forespore membrane where it functions to maintain the integrity of the CotE ring and to anchor the nascent coat to the underlying spore structures. However, it is not known which spore coat proteins interact directly with SpoVID. In this study we examined the interaction between SpoVID and another spore coat protein, SafA, in vivo using the yeast two-hybrid system and in vitro. We found evidence that SpoVID and SafA directly interact and that SafA interacts with itself. Immunofluorescence microscopy showed that SafA localized around the forespore early during coat assembly and that this localization of SafA was dependent on SpoVID. Moreover, targeting of SafA to the forespore was also dependent on SpoIVA, as was targeting of SpoVID to the forespore. We suggest that the localization of SafA to the spore coat requires direct interaction with SpoVID.Proteins are targeted to specific subcellular locations during the assembly of a variety of bacterial structures. The structures assembled by prokaryotes include, for example, the cell division septum (3), surface protein layers (S-layers) (36), and a number of surface appendages such as flagella (24) and pili (17). Here we are concerned with the assembly of the Bacillus subtilis spore coat, a proteinaceous structure that encases spores (reviewed in references 7 and 16). The B. subtilis spore is a metabolically dormant cell type that is formed as an adaptive response to nutrient depletion (9, 30, 37). The spore coat provides protection against physical and chemical insults and can sense and respond to nutrients that trigger germination (9, 26).Spore morphogenesis commences by the placement of an asymmetric septum that divides the rod-shaped cell into a larger mother cell and a smaller prespore, each containing a copy of the chromosome. The septal membranes migrate around the prespore, in a process known as engulfment, which eventually results in the formation of a free-floating protoplast (the forespore) separated from the mother cell cytoplasm by a double-membrane system. Cell wall material (the cortex) is deposited between the membranes of the forespore (9, 30, 37). A thick coat composed of more than two dozen proteins is assembled around the outer forespore membrane. The majority of the proteins that make up the coat are synthesized in the mother cell under the direction of mother cell-specific RNA polymerase sigma factors (7,16,30).