Ingrid C. Deckman

Physical studies of ribosomal protein-RNA interactions.

Publisher Summary This chapter presents physical studies of ribosomal protein–RNA interactions. The chapter deals with r-protein S4 binding a specific site on α mRNA, but should be applicable to other r-protein interactions with rRNA or mRNA fragments. The filter retention assay is useful, but one has to be aware that (1) specific and nonspecific complexes of protein with the same RNA may be detected with widely different efficiencies, and (2) the level of competing nonspecific binding may change dramatically as the endpoints of the RNA fragment change. The sucrose gradient assay described here is probably easier to interpret, as it depends only on the reequilibration of protein and protein-RNA complexes during the time of the sedimentation run. Although the absolute affinities measured may be systematically off by a factory of two or three, changes in affinity with RNA sequence or buffer composition should be accurately detected. The relative affinity of a small RNA fragment and a large RNA for a protein should also be measured accurately in competition experiments. The simplicity of the sucrose gradient assays and the unambiguous interpretation of the results should make them a useful complement to filter binding assays for quantitative studies.

S4-α mRNA translation repression complex: I. Thermodynamics of formation

Abstract Expression of the four ribosomal proteins from the Escherichia coli α operon (S4, S11, S13, and L17) is regulated at the level of translation by the binding of S4 to the α mRNA. Using a filter binding assay and α mRNA sequences prepared by in-vitro transcription, previous work located the S4 target site within the ~ 100-base leader sequence. We have extended this work to include fragments of the α leader with six different 5′ end points and four different 3′ end points. A core region between bases 23 and 69 (numbering from the first nucleotide of the E. coli transcript) binds S4 with an affinity of ~ 2 μ m −1. Regions of weak interactions are located in the 22 nucleotides 5′ and the 70 nucleotides 3′ to this core; they increase the S4 affinity to ~ 13μ m −1. Studies of S4-α mRNA binding under different conditions have revealed the following. (1) Specific and non-specific binding show the same dependence on K+ concentration, with ∂ log K ∂ log [K + ] ≈ 4 in most potassium salts. With KC1 and KBr, much weaker salt dependence of specific complex formation is observed suggesting that the protein responds to the correct RNA substrate by binding halide anions. (2) Increasing the MgCl2 concentration between 1 and 4 m m enhances binding by a factor of 4, with no further effects up to 20 m m . About five Mg2+ are taken up by the complex with an average binding constant of ~ 600 m −1 each. Renaturation of the RNA in the presence of MgCl2 is also required to obtain full binding. These effects are seen only with α mRNA extending beyond the initiation codon; S4 binding to the α leader sequence itself is insensitive to Mg2+. (3) The association kinetics are fast and probably diffusion controlled. (4) Formation of the complex is entirely entropy driven.

Structural Basis for Elastolytic Substrate Specificity in Rodent α-Chymases

Divergence of substrate specificity within the context of a common structural framework represents an important mechanism by which new enzyme activity naturally evolves. We present enzymological and x-ray structural data for hamster chymase-2 (HAM2) that provides a detailed explanation for the unusual hydrolytic specificity of this rodent α-chymase. In enzymatic characterization, hamster chymase-1 (HAM1) showed typical chymase proteolytic activity. In contrast, HAM2 exhibited atypical substrate specificity, cleaving on the carboxyl side of the P1 substrate residues Ala and Val, characteristic of elastolytic rather than chymotryptic specificity. The 2.5-Å resolution crystal structure of HAM2 complexed to the peptidyl inhibitor MeOSuc-Ala-Ala-Pro-Ala-chloromethylketone revealed a narrow and shallow S1 substrate binding pocket that accommodated only a small hydrophobic residue (e.g. Ala or Val). The different substrate specificities of HAM2 and HAM1 are explained by changes in four S1 substrate site residues (positions 189, 190, 216, and 226). Of these, Asn189, Val190, and Val216 form an easily identifiable triplet in all known rodent α-chymases that can be used to predict elastolytic specificity for novel chymase-like sequences. Phylogenetic comparison defines guinea pig and rabbit chymases as the closest orthologs to rodent α-chymases.

A novel fluorogenic substrate for the measurement of endothelial lipase activity

Endothelial lipase (EL) is a phospholipase A1 (PLA1) enzyme that hydrolyzes phospholipids at the sn-1 position to produce lysophospholipids and free fatty acids. Measurement of the PLA1 activity of EL is usually accomplished by the use of substrates that are also hydrolyzed by lipases in other subfamilies such as PLA2 enzymes. In order to distinguish PLA1 activity of EL from PLA2 enzymatic activity in cell-based assays, cell supernatants, and other nonhomogeneous systems, a novel fluorogenic substrate with selectivity toward PLA1 hydrolysis was conceived and characterized. This substrate was preferred by PLA1 enzymes, such as EL and hepatic lipase, and was cleaved with much lower efficiency by lipases that exhibit primarily triglyceride lipase activity, such as LPL or a lipase with PLA2 activity. The phospholipase activity detected by the PLA1 substrate could be inhibited with the small molecule esterase inhibitor ebelactone B. Furthermore, the PLA1 substrate was able to detect EL activity in human umbilical vein endothelial cells in a cell-based assay. This substrate is a useful reagent for identifying modulators of PLA1 enzymes, such as EL, and aiding in characterizing their mechanisms of action.

The HIV-1 Aspartyl Protease: Maturation and Substrate Specificity

In all retroviruses, the viral aspartyl protease is first translated as part of a large polyprotein precursor, the 160 kilodalton gag-pol polyprotein in the case of HIV-1 (Rey et al, 1987; Jacks et al, 1988). Experimental evidence strongly suggests that HIV-1 viral particles initially bud out of the host cell with an immature structure and composition, containing unprocessed PrSSgag and Pr160gag-Pol polyproteins (Hockley et al, 1988; Gottlinger et al, 1989, Peng et al, 1989). The question of when and how the protease actually processes these polyproteins is important for the understanding of the viral life cycle but remains to be answered. In an effort to gain information on this process, we undertook a series of genetic studies in Escherichia coli on the maturation of the protease from its precursor forms and on this protease substrate preferences. This information should not only contribute to our understanding of the viral maturation process, but also assist us in the design of inhibitors of this essential viral enzyme.