Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0162473 (Frey)
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In the structural protein open reading frame (SP-ORF) of rubella virus (RUB), the sequences for the three virion proteins occur in the order NH2-C-E2-E1-COOH with hydrophobic, consensus signal sequences preceding the amino termini for each of the two membrane proteins (T. K. Frey and L. D. Marr, 1988 Gene 62, 85-100). In vitro translation in the presence of microsomes of RNA transcripts from a plasmid containing the SP-ORF resulted in production and accurate processing of the three structural proteins. Since in the absence of microsomes the 110-kDa precursor of these proteins is produced, this finding indicated that the cleavage events in processing of the precursor were mediated by signalase. To study the C-E2 processing event, a DNA construct was made which contained the sequences for E2 beginning at the NH2 terminus of the hydrophobic consensus signal and extending through to the NH2 terminus of E1. In vitro translation of transcripts from this construct in the presence of microsomes resulted in accurate processing of E2 confirming that the hydrophobic sequence was a signal sequence and demonstrating it could function externally as well as internally within the 110-kDa precursor. To determine if the E2 signal was maintained on C after cleavage of the precursor by signalase, the SP-ORF plasmid was mutagenized to place translation termination codons at either the NH2 or COOH side of the E2 signal sequence such that C protein lacking or containing the E2 signal would be produced. As expected, the C-minus-signal protein migrated more rapidly in polyacrylamide gels than did the C-plus-signal protein. C translated from the SP-ORF construct as well as authentic C from infected cells comigrated with the C-plus-signal protein, indicating that the E2 signal was not removed. In a corollary study, it was found that RUB C protein was phosphorylated in vivo, although the percentage of the protein phosphorylated was not determined.
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PMID:Efficient in vitro translation and processing of the rubella virus structural proteins in the presence of microsomes. 198 59

The sequence of the 3' 4508 nucleotides (nt) of the genomic RNA of the Therien strain of rubella virus (RV) was determined for cDNA clones. The sequence contains a 3189-nt open reading frame (ORF) which codes for the structural proteins C, E2 and E1. C is predicted to have a length of 300 amino acids (aa). The N-terminal half of the C protein is highly basic and hydrophilic in nature, and is putatively the region of the protein which interacts with the virion RNA. At the C terminus of the C protein is a stretch of 20 hydrophobic aa which also serves as the signal sequence for E2, indicating that the cleavage of C from the polyprotein precursor may be catalyzed by signalase in the lumen of the endoplasmic reticulum. E2 is 282 aa in length and contains four potential N-linked glycosylation sites and a putative transmembrane domain near its C terminus. The sequence of E1 has been previously described [Frey et al., Virology 154 (1986) 228-232]. No homology could be detected between the amino acid sequence of the RV structural proteins and the amino acid sequence of the alphavirus structural proteins. From the position of a region of 30 nt in the RV genomic sequence which exhibited significant homology with the sequence in the alphavirus genome at which subgenomic RNA synthesis is initiated, the RV subgenomic RNA is predicted to be 3346 nt in length and the nontranslated region from the 5' end of the subgenomic RNA to the structural protein ORF is predicted to be 98 nt. In a different translation frame beginning at the 5' end of the RV nt sequence reported here is a 1407 nt ORF which is the C terminal region of the nonstructural protein ORF. This ORF overlaps the structural protein ORF by 149 nt. A low level of homology could be detected between the predicted amino acid sequence of the C-terminus of the RV nonstructural protein ORF and the replicase proteins of several positive RNA viruses of animals and plants, including nsp4 of the alphaviruses, the protein encoded by the C-terminal region of the alphavirus nonstructural ORF. However, the overall homology between RV and the alphaviruses in this region of the genome was only 18%, indicating that these two genera of the Togavirus family are only distantly related. Intriguingly, there is a 2844-nt ORF present in the negative polarity orientation of the RV sequence which could encode a 928-aa polyprotein.
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PMID:Sequence of the region coding for virion proteins C and E2 and the carboxy terminus of the nonstructural proteins of rubella virus: comparison with alphaviruses. 283 71

We report here the x-ray crystal structure of a soluble catalytically active fragment of the Escherichia coli type I signal peptidase (SPase-(Delta2-75)) in the absence of inhibitor or substrate (apoenzyme). The structure was solved by molecular replacement and refined to 2.4 A resolution in a different space group (P4(1)2(1)2) from that of the previously published acyl-enzyme inhibitor-bound structure (P2(1)2(1)2) (Paetzel, M., Dalbey, R.E., and Strynadka, N.C.J. (1998) Nature 396, 186-190). A comparison with the acyl-enzyme structure shows significant side-chain and main-chain differences in the binding site and active site regions, which result in a smaller S1 binding pocket in the apoenzyme. The apoenzyme structure is consistent with SPase utilizing an unusual oxyanion hole containing one side-chain hydroxyl hydrogen (Ser-88 OgammaH) and one main-chain amide hydrogen (Ser-90 NH). Analysis of the apoenzyme active site reveals a potential deacylating water that was displaced by the inhibitor. It has been proposed that SPase utilizes a Ser-Lys dyad mechanism in the cleavage reaction. A similar mechanism has been proposed for the LexA family of proteases. A structural comparison of SPase and members of the LexA family of proteases reveals a difference in the side-chain orientation for the general base lysine, both of which are stabilized by an adjacent hydroxyl group. To gain insight into how signal peptidase recognizes its substrates, we have modeled a signal peptide into the binding site of SPase. The model is built based on the recently solved crystal structure of the analogous enzyme LexA (Luo, Y., Pfuetzner, R. A., Mosimann, S., Paetzel, M., Frey, E. A., Cherney, M., Kim, B., Little, J. W., and Strynadka, N. C. J. (2001) Cell 106, 1-10) with its bound cleavage site region.
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PMID:Crystal structure of a bacterial signal peptidase apoenzyme: implications for signal peptide binding and the Ser-Lys dyad mechanism. 1174 64