Crystal structure of the ribonucleoprotein core of the signal recognition particle.

The signal recognition particle (SRP), a protein-RNA complex conserved in all three kingdoms of life, recognizes and transports specific proteins to cellular membranes for insertion or secretion. We describe here the 1.8 angstrom crystal structure of the universal core of the SRP, revealing protein recognition of a distorted RNA minor groove. Nucleotide analog interference mapping demonstrates the biological importance of observed interactions, and genetic results show that this core is functional in vivo. The structure explains why the conserved residues in the protein and RNA are required for SRP assembly and defines a signal sequence recognition surface composed of both protein and RNA.

Large-scale purification of a stable form of recombinant tobacco etch virus protease.

Tobacco etch virus NIa proteinase (NIa-Pro) has become the enzyme of choice for removing tags and fusion domains from recombinant proteins in vitro. We have designed a mutant NIa-Pro that resists autoproteolytic inactivation and present an efficient method for producing large amounts of this enzyme that is highly pure, active, and stable over time. Histidine-tagged forms of both wild-type and mutant NIa-Pro were overexpressed in E. coli under conditions in which greater than 95% of the protease was in the insoluble fraction after cell lysis. An inclusion body preparation followed by denaturing purification over a single affinity column and protein renaturation yields greater than 12.5 mg enzyme per liter of bacterial cell culture. NIa-Pro purified according to this protocol has been used for quantitative removal of fusion domains from a variety of proteins prepared for crystallization and biochemical analysis.

Role of the hemA gene product and delta-aminolevulinic acid in regulation of Escherichia coli heme synthesis.

We initiated these studies to help clarify the roles of heme, delta-aminolevulinic acid (ALA), hemA, and hemM in Escherichia coli heme synthesis. Using recombinant human hemoglobin (rHb1.1) as a tool for increasing E. coli's heme requirements, we demonstrated that heme is a feedback inhibitor of heme synthesis. Cooverexpression of rHb1.1 and the hemA-encoded glutamyl-tRNA (GTR) reductase increased intracellular levels of ALA and heme and increased the rate of rHb1.1 formation. These results support the conclusion that heme synthesis is limited by ALA (S. Hino and A. Ishida, Enzyme 16:42-49, 1973; W. K. Philipp-Dormston and M. Doss, Enzyme 16:57-64, 1973) and that the hemA-encoded GTR reductase is a rate-limiting enzyme in the pathway (J.-M. Li, C. S. Russell, and S. D. Cosloy, Gene 82:2099-217, 1989). Increasing the copy number of hemM, whose product is believed to be required for efficient ALA formation (W. Chen, C. S. Russell, Y. Murooka, and S. D. Cosloy, J. Bacteriol. 176:2743-2746, 1994; M. Ikemi, K. Murakami, M. Hashimoto, and Y. Murooka, Gene 121:127-132, 1992), had no effect on either ALA pools or the rate of rHb1.1 accumulation. The hemA-encoded GTR reductase was found to be regulated by ALA. Some of our results differ from those reported by Hart and coworkers (R. A. Hart, P. T. Kallio, and J. E. Bailey, Appl. Environ. Microbiol. 60:2431-2437, 1994), who concluded that ALA formation is not the rate-limiting step in E. coli cells expressing Vitreoscilla hemoglobin.