The binding of signal recognition particle (SRP) to ribosome-bound signal sequences has been characterized directly and quantitatively using fluorescence spectroscopy. A fluorescent probe was incorporated cotranslationally into the signal sequence of a ribosome⅐nascent chain complex (RNC), and upon titration with SRP, a large and saturable increase in fluorescence intensity was observed. Spectral analyses of SRP and RNC association as a function of concentration allowed us to measure, at equilibrium, K d values of 0.05-0.38 nM for SRP⅐RNC complexes with different signal sequences. Competitive binding experiments with nonfluorescent RNC species revealed that the nascent chain probe did not alter SRP affinity and that SRP has significant affinity for both nontranslating ribosomes (K d ؍ 71 nM) and RNCs that lack an exposed signal sequence (K d ؍ 8 nM). SRP can therefore distinguish between translating and nontranslating ribosomes. The very high signal sequence-dependent SRP⅐RNC affinity did not decrease as the nascent chain lengthened. Thus, the inhibition of SRPdependent targeting of RNCs to the endoplasmic reticulum membrane observed with long nascent chains does not result from reduced SRP binding to the signal sequence, as widely thought, but rather from a subsequent step, presumably nascent chain interference of SRP⅐RNC association with the SRP receptor and/or translocon.In mammalian cells, ribosomes are found both in the cytoplasm and at the membrane of the endoplasmic reticulum (ER).1 These two classes of ribosome differ in the nature of their translation products, with membrane-bound ribosomes synthesizing secretory or membrane proteins that are being translocated across or integrated into the ER membrane cotranslationally. The structural feature that distinguishes the cytoplasmic ribosomes from the membrane-bound ribosomes is the presence in the latter of a nascent chain that contains a signal sequence. When a nascent chain signal sequence emerges from the ribosome, it is recognized and bound by a ribonucleoprotein termed the signal recognition particle (SRP) (for review, see Ref. 1). The binding of SRP to the signal sequence-containing ribosome-nascent chain complex (RNC) temporarily prevents it from synthesizing protein.The resulting "elongation-arrested complex" then diffuses to the ER membrane where a GTP-dependent interaction with the SRP receptor initiates a series of events which includes the binding of the RNC to the site of cotranslational translocation and integration at the ER membrane (the translocon), the release of the signal sequence from the SRP, the release of SRP and the SRP receptor from the translocon, and the resumption of protein synthesis by the ribosome (for review, see Refs. 1-3). After targeting is complete, nascent chain translocation or integration then proceeds at the translocon (for review, see Ref. 4). SRP therefore has a critical regulatory role in the cell because it is responsible both for the proper trafficking of newly synthesized proteins and for the conversion of ...
Normal endocrine development and function require nuclear hormone receptor SF-1 (steroidogenic factor 1). To understand the molecular mechanism of SF-1 action, we have investigated its domain function by mutagenesis and functional analyses. Our mutant studies show that the putative AF2 (activation function 2) helix located at the C-terminal end is indispensable for gene activation. SF-1 does not have an N-terminal AF1 domain. Instead, it contains a unique FP region, composed of the Ftz-F1 box and the proline cluster, after the zinc finger motif. The FP region interacts with transcription factor IIB (TFIIB) in vitro. This interaction requires residues 178-201 of TFIIB, a domain capable of binding several transcription factors. The FP region also mediates physical interaction with c-Jun, and this interaction greatly enhances SF-1 activity. The putative SF-1 ligand, 25-hydroxycholesterol, has no effects on these bindings. In addition, the Ftz-F1 box contains a bipartite nuclear localization signal (NLS). Removing the basic residues at either end of the key nuclear localization sequence NLS2.2 abolishes the nuclear transport. Expression of mutants containing only the FP region or lacking the AF2 domain blocks wild-type SF-1 activity in cells. By contrast, the mutant having a truncated nuclear localization signal lacks this dominant negative effect. These results delineate the importance of the FP and AF2 regions in nuclear localization, protein-protein interaction, and transcriptional activation.
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