The sequence and spacing requirements of the archaeal "distal promoter element' (DPE) were examined by randomizing positions -19 to -32 upstream of the transcriptional start site of the ferredoxin (fdx) promoter of Halobacterium salinarium. This randomized promoter library containing 4(14) entries was cloned in front of the dihydrofolate reductase (DHFR) reporter gene and transformed into Haloferax volcanii. Two approaches were used to characterize these synthetic promoters. First, 1040 independent clones were randomly chosen and their degrees of trimethoprim resistance were determined. The sequences of 20 clones that were either sensitive, partially resistant or very resistant, respectively, were determined. Secondly, the transformed library was screened by direct selection for high-activity promoters by growing transformants in the presence of trimethoprim. Both approaches produced the following consensus sequence for a halobacterial promoter: (Formula: see text) (where R = A or G; Y = C or T; W = A or T; S = G or C; N = A, C, G or T). Further characterization of two sensitive, two partially resistant, and two very resistant clones verified that DHFR activity and cell phenotype are directly correlated. Sensitive clones did not contain detectable dhfr mRNA, whereas partially resistant clones contained a 700 nucleotide (nt)-long transcript, and very resistant clones contained both the 700nt-long transcript and a second, more abundant, 500nt-long truncated transcript. Quantification of the dhfr mRNA and DHFR enzyme activity suggests that the 3'-untranslated region of the dhfr transcript, missing from the shorter transcript, functions as a negative regulator of translation.
RNA-binding proteins are central to posttranscriptional gene regulation and play an important role in a number of major human diseases. Cloning such proteins is a crucial but often difficult step in elucidating the biological function of RNA regulatory elements. To make it easier to clone proteins that specifically bind RNA elements of interest, we have developed a rapid and broadly applicable in vitro genetic selection method based on T7 phage display. Using hairpin II of U1 small nuclear RNA (U1hpII) or the 3 stem loop of histone mRNA as bait, we could selectively amplify T7 phage that display either the spliceosomal protein U1A or the histone stem loop-binding protein from a lung cDNA phage library containing more than 10 7 independent clones. The use of U1hpII mutants with various affinities for U1A revealed that this method allows the selection even of proteins that bind their cognate RNA targets with relatively weak affinities (Kd as high as the micromolar range). Experiments with a mixture of recombinant phage displaying U1A or the closely related protein U2B؆ demonstrated that addition of a competitor RNA can suppress selection of a protein with a higher affinity for a given RNA target, thereby allowing the preferential amplification of a lower affinity protein. Together, these findings suggest that T7 phage display can be used to rapidly and selectively clone virtually any protein that binds a known RNA regulatory element, including those that bind with low affinity or that must compete for binding with other proteins. R NA-binding proteins (RNA-BPs) play a key role in a variety of posttranscriptional regulatory processes, including RNA processing, nucleocytoplasmic transport, translation, and mRNA decay (1-3). Moreover, a burgeoning body of evidence has implicated a number of RNA-BPs in the genetic etiology of human diseases such as fragile X mental retardation (4), paraneoplastic neurologic disorders (5), and spinal muscular atrophy (6), as well as in many microbial infections, including AIDS (7) and influenza (8).An essential step in elucidating the regulatory mechanism of a given RNA element is to identify and characterize the protein(s) that binds to this element and mediates its effect. Characterization of such an RNA-BP is most readily accomplished by cloning its cDNA. Traditionally, this has been achieved by protein purification, peptide microsequencing, and cDNA amplification with PCR primers designed on the basis of the amino acid sequence information. This labor-intensive strategy requires an enormous effort and can be particularly difficult in the case of RNA-BPs present at a low cellular concentration.Some genetic screening methods have been developed to facilitate the cloning of RNA-BP cDNAs, most notably the yeast three-hybrid system (9, 10) and plaque-lift analysis (11,12). Both techniques have allowed the cloning of previously unidentified RNA-BPs (13-16). However, several limitations may interfere with the general applicability of these procedures. For example, when using the three-hyb...
Phosducin is a 33-kDa cytosolic regulator of G-protein-mediated signaling that has previously been thought to be specific for retina and pineal gland. In this study, we show widespread tissue distribution of phosducin by the amplification of its cDNA and the detection of two different transcripts in Northern analyses in liver, lung, heart, brain, and retina. On the protein level, phosducin could be detected in 12 bovine tissues by immune precipitation and subsequent Western analysis using anti-phosducin antibodies generated in two different species. Masking of phosducin in direct Western blots appears to explain the failure to detect phosducin in earlier studies. The concentration of phosducin in bovine brain was calculated in the range of 10 pmol/mg total cytosolic protein (-1I,uM), whereas in the other tissues, it was slightly less. In these concentrations, phosducin inhibited receptor-stimulated adenylyl cyclase activity in cell membranes by about 50%. Taken together, our results indicate that phosducin is a ubiquitous regulator of G-protein function.GTP-binding proteins (G proteins) couple many kinds of cell surface receptors to intracellular effectors or ion channels and are thereby crucial for the specificity, velocity, and amplification of signal transduction across the cell membrane (1, 2). Agonist-occupied receptors stimulate the binding of GTP to the a subunits of G proteins (Ga), which, in turn, promotes the dissociation of the G protein 13y (Gf3y) subunits, allowing both the Ga and GfBy subunits to exert their various specific functions (3). These transduction cascades are regulated by multiple mechanisms, which have been investigated most thoroughly for the j3-adrenergic receptor/stimulatory G protein (Gs)/adenylyl cyclase (4) and the rhodopsin/transducin (Gt)/cGMP phosphodiesterase (5) systems. Regulation occurs predominantly at the receptor level but also at the level of G proteins themselves.Phosducin is a 33-kDa soluble phosphoprotein present in the retina (6, 7) and the developmentally related pineal gland (8, 9). It forms complexes with the P3'y subunits of transducin, the retinal G protein (6). The Gf3y binding site has been mapped to its N terminus (10, 11). Phosducin affects G-proteinmediated signal transduction by high-affinity binding to Gf3y and may, thereby, regulate the number of f3y subunits available to interact with the Ga subunits. Phosducin has been shown to inhibit the intrinsic GTPase activity of Gs, and inhibitory and "other" G proteins (G1 and Go, respectively) equally well (12). Similarly, it has been shown to reduce the activity of Gt (13 This study was undertaken to test this hypothesis and to investigate the presence of phosducin and its mRNA in nonretinal/pineal tissues. We demonstrate that it occurs in a wide variety of bovine tissues at the mRNA level and protein level, respectively, and that its concentration in these tissues is sufficient to act as a G-protein regulator. MATERIALS AND METHODSRNA Isolation and Northern Blot Analysis. Total RNA was isolated from...
Prolonged agonist stimulation of  2 -adrenergic receptors results in receptor down-regulation, which is closely associated with a reduction of the corresponding mRNA, an effect mediated in part by changes in mRNA stability. Transfection experiments with human  2 -adrenergic receptor cDNAs bearing or lacking the untranslated regions suggested that the essential agonist sensitivity of the mRNA resides within the 3-untranslated region. The importance of this region was further confirmed in gel shift experiments; cytosolic preparations from agonist-stimulated DDT 1 -MF2 smooth muscle cells caused a shift of  2 -adrenergic receptor mRNAs containing the 3-untranslated region. Progressive 3-terminal truncations of the receptor cDNA led to the identification of an AU-rich element at positions 329 -337 of the 3-untranslated region as the responsible cisacting element. Substitution of this motif by cytosine residues almost completely abolished mRNA down-regulation and inhibited the formation of the RNA-protein complex. Even though the  2 -adrenergic receptor AUrich element showed two U 3 A transitions compared with the recently proposed AU-rich element consensus sequence, it revealed an almost identical destabilizing potency. Fusion of the  2 -adrenergic receptor 3-untranslated region to the -globin coding sequence dramatically reduced the half-life of the chimeric transcript in an agonist-and cAMP-dependent manner. This suggests that the agonist-induced  2 -adrenergic receptor mRNA destabilization is regulated by cAMP-dependent RNA-binding protein(s) via a specific AU-rich element.Chronic stimulation of the  2 -adrenergic receptor ( 2 AR) 1 results in a decrease of receptor responsiveness, a process called agonist-induced receptor desensitization (1, 2). Long term desensitization often involves a significant reduction of receptor numbers, which is termed receptor down-regulation. Several distinct molecular mechanisms affecting both mRNA and protein levels contribute to receptor down-regulation (2-4), which appear to be operative to varying extents in different cell lines. To date there is evidence that the expression of the  2 AR gene can be regulated at the level of transcription (5, 6), posttranscriptionally at the level of mRNA stability (7) or at the level of translation via a short peptide encoded within the 5Ј-untranslated region (UTR) of the  2 AR gene (8).Posttranscriptional mechanisms are of particular interest, since they participate in the stability and turnover of various highly labile mRNAs, such as granulocyte-macrophage colonystimulating factor, interleukin-3, and the oncogenes c-fos and c-myc (9, 10). AU-rich elements (AREs) are often found in the 3Ј-UTRs of these mRNAs and appear to be key determinants of their short half-lives, even if mRNA turnover does not strictly depend on these motifs. The optimal destabilization motif was recently suggested to be UUAUUUA(U/A)(U/A) (11, 12), but there is also evidence that an AUUUA pentamer need not be an integral part of a functional ARE (13). On the co...
Phosducin, which tightly binds ␥-subunits of heterotrimeric G-proteins, has been conjectured to play a role in regulating second messenger signaling cascades, but to date its specific function has not been elucidated. Here we demonstrate a potential role for phosducin in regulating olfactory signal transduction. In isolated olfactory cilia certain odorants elicit a rapid and transient cAMP response, terminated by a concerted process which requires the action of two protein kinases, protein kinase A (PKA) and a receptor-specific kinase (GRK3) (Schleicher, S., Boekhoff, I. Arriza, J., Lefkowitz, R. J., and Breer, H. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 1420 -1424). The mechanism of action of GRK3 involves a G␥-mediated translocation of the kinase to the plasma membrane bound receptors (Pitcher, J. A., Inglese, J., Higgins, J. B., Arriza, J. L., Casey, P. J., Kim, C., Benovic, J. L., Kwatra, M. M., Caron, M. G., and Lefkowitz, R. J. (1992) Science 257, 1264 -1267). A protein with a molecular mass of 33 kDa that comigrates on SDS gels with recombinant phosducin and which is immunoreactive with phosducin antibodies is present in olfactory cilia. Recombinant phosducin added to permeabilized olfactory cilia preparations strongly inhibits termination of odorant-induced cAMP response and odorantinduced membrane translocation of GRK3. In addition, the cAMP analogue dibutyryl cAMP stimulates membrane targeting of the receptor kinase. This effect is presumably due to PKA-mediated phosphorylation of phosducin, which diminishes its affinity for binding to the G␥-subunit, thereby making G␥ available to function as a membrane anchor for GRK3. A specific PKA inhibitor blocks the odorant-induced translocation of the receptor kinase. Consistent with this formulation, a non-phosphorylatable mutant of phosducin (phosducin Ser-73 3 Ala) is an even more effective inhibitor of desensitization and membrane targeting of GRK3 than the wild-type protein. A phosducin mutant that mimics phosphorylated phosducin (phosducin Ser-73 3 Asp) lacks this property and in fact recruits GRK3 to the membrane and potentiates desensitization. These results suggest that phosducin may act as a phosphorylation-dependent switch in second messenger signaling cascades, regulating the kinetics of desensitization processes by controlling the activity of G␥-dependent GRKs.The olfactory system responds precisely to iterative stimulation; this characteristic feature is due to the phasic responses of receptor cells (3), based on a rapid termination of the odorinduced primary reaction (4). Recent studies have indicated that olfactory signaling is terminated by uncoupling the transduction cascade; the second messenger signal elicited by odors is turned off by a negative feedback reaction controlled by phosphorylation of odorant receptors mediated by two types of enzymes, a second messenger controlled kinase and a receptor specific kinase (GRK) 1 (1, 5-8). These observations raise the possibility that the two kinases act sequentially in a reaction cascade tha...
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