A chloroplast processing enzyme involved in precursor maturation shares a zinc-binding motif with a recently recognized family of metalloendopeptidases.

Nuclear-encoded proteins targeted to the chloroplast are typically synthesized with N-terminal transit peptides which are proteolytically removed upon import. Structurally related proteins of 145 and 143 kDa copurify with a soluble chloroplast processing enzyme (CPE) that cleaves the precursor for the major light-harvesting chlorophyll a/b binding protein and have been implicated in the maturation of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase and acyl carrier protein. The 145- and 143-kDa proteins have not been found as a heterodimer and thus may represent functionally independent isoforms encoded by separate genes. Here we describe the primary structure of a 140-kDa polypeptide encoded by cDNAs isolated by using antibodies raised against the 145/143-kDa doublet. The 140-kDa polypeptide contains a transit peptide, and strikingly, a His-Xaa-Xaa-Glu-His zinc-binding motif that is conserved in a recently recognized family of metalloendopeptidases, which includes Escherichia coli protease III, insulin-degrading enzyme, and subunit beta of the mitochondrial processing peptidase. Identity of 25-30%, concentrated near the N terminus of the 140-kDa polypeptide, is found with these proteases. Expression of CPE in leaves is not light dependent. Indeed, transcripts are present in dark-grown plants, and the 145/143-kDa doublet and proteolytic activity are both found in etioplasts, as well as in root plastids. Thus, CPE appears to be a necessary component of the import machinery in photosynthetic and nonphotosynthetic tissues, and it may function as a general stromal processing peptidase in plastids.

The tRNA processing enzyme RNase T is essential for maturation of 5S RNA.

The maturation of 5S RNA in Escherichia coli is poorly understood. Although it is known that large precursors of 5S RNA accumulate in mutant cells lacking the endoribonuclease-RNase E, almost nothing is known about how the mature 5' and 3' termini of these molecules are generated. We have examined 5S RNA maturation in wild-type and single- or multiple-exoribonuclease-deficient cells by Northern blot and primer-extension analysis. Our results indicate that no mature 5S RNA is made in RNase T-deficient strains. Rather, 5S RNA precursors containing predominantly 2 extra nucleotides at the 3' end accumulate. Apparently, these 5S RNAs are functional inasmuch as mutant cells are viable, growing only slightly slower than wild type. Purified RNase T can remove the extra 3' residues, showing that it is directly involved in the trimming reaction. In contrast, mutations affecting other 3' exoribonucleases have no effect on 5S RNA maturation. Approximately 90% of the 5S RNAs in both wild-type and RNase T- cells contain mature 5' termini, indicating that 5' processing is independent of RNase T action. These data identify the enzyme responsible for generating the mature 3' terminus of 5S RNA molecules and also demonstrate that a completely processed 5S RNA molecule is not essential for cell survival.

Image processing via level set curvature flow.

We present a controlled image smoothing and enhancement method based on a curvature flow interpretation of the geometric heat equation. Compared to existing techniques, the model has several distinct advantages. (i) It contains just one enhancement parameter. (ii) The scheme naturally inherits a stopping criterion from the image; continued application of the scheme produces no further change. (iii) The method is one of the fastest possible schemes based on a curvature-controlled approach.