Amino acid toxicities of Escherichia coli that are prevented by leucyl-tRNA synthetase amino acid editing.

Leucyl-tRNA synthetase (LeuRS) has evolved an editing function to clear misactivated amino acids. An Escherichia coli-based assay was established to identify amino acids that compromise the fidelity of LeuRS and translation. Multiple nonstandard as well as standard amino acids were toxic to the cell when LeuRS editing was inactivated.

A glycine hinge for tRNA-dependent translocation of editing substrates to prevent errors by leucyl-tRNA synthetase.

Aminoacyl-tRNA synthetases often rely on a proofreading mechanism to clear mischarging errors before they can be incorporated into newly synthesized proteins. Leucyl-tRNA synthetase (LeuRS) houses a hydrolytic editing pocket in a domain that is distinct from its aminoacylation domain. Mischarged amino acids are transiently translocated approximately 30A between active sites for editing by an unknown tRNA-dependent mechanism. A glycine within a flexible beta-strand that links the aminoacylation and editing domains of LeuRS was determined to be important to tRNA translocation. The translocation-defective mutation also demonstrated that the editing site screens both correctly and incorrectly charged tRNAs prior to product release.

The capsid protein of human immunodeficiency virus: interactions of HIV-1 capsid with host protein factors.

HIV-1 is a retrovirus that causes AIDS in humans. The RNA genome of the virus encodes a Gag polyprotein, which is further processed into matrix, capsid and nucleocapsid proteins. These proteins play a significant role at several steps in the viral life cycle. In addition, various stages of assembly, infection and replication of the virus involve necessary interactions with a large number of supplementary proteins/cofactors within the infected host cell. This minireview focuses on the proteomics of the capsid protein, its influence on the packaging of nonviral molecules into HIV-1 virions and the subsequent role of the molecules themselves. These interactions and their characterization present novel frontiers for the design and advancement of antiviral therapeutics.

Functional segregation of a predicted "hinge" site within the beta-strand linkers of Escherichia coli leucyl-tRNA synthetase.

Some aminoacyl-tRNA synthetases (AARSs) employ an editing mechanism to ensure the fidelity of protein synthesis. Leucyl-tRNA synthetase (LeuRS), isoleucyl-tRNA synthetase (IleRS), and valyl-tRNA synthetase (ValRS) share a common insertion, called the CP1 domain, which is responsible for clearing misformed products. This discrete domain is connected to the main body of the enzyme via two beta-strand tethers. The CP1 hydrolytic editing active site is located approximately 30 A from the aminoacylation active site in the canonical core of the enzyme, requiring translocation of mischarged amino acids for editing. An ensemble of crystal and cocrystal structures for LeuRS, IleRS, and ValRS suggests that the CP1 domain rotates via its flexible beta-strand linkers relative to the main body along various steps in the enzyme's reaction pathway. Computational analysis suggested that the end of the N-terminal beta-strand acted as a hinge. We hypothesized that a molecular hinge could specifically direct movement of the CP1 domain relative to the main body. We introduced a series of mutations into both beta-strands in attempts to hinder movement and alter fidelity of LeuRS. Our results have identified specific residues within the beta-strand tethers that selectively impact enzyme activity, supporting the idea that beta-strand orientation is crucial for LeuRS canonical core and CP1 domain functions.