DNA stimulates ATP-dependent proteolysis and protein-dependent ATPase activity of protease La from Escherichia coli.

Abstract

The product of the Ion gene in Escherichia coli is an ATP-dependent protease, protease La, that also binds strongly to DNA. Addition of double-stranded or single-stranded DNA to the protease in the presence ofATP was found to stimulate the hydrolysis of casein or globin 2-to 7-fold, depending on the DNA concentration. Native DNA from several sources (plasmid pBR322, phage T7, or calf thymus) had similar effects, but after denaturation the DNA was 20-100% more effective than the native form. Although poly(rA), globin mRNA, and various tRNAs did not stimulate proteolysis, poly(rC) and poly(rU) were effective.Poly(dT) was stimulatory but (dT)1o was not. In the presence of DNA as in its absence, proteolysis required concomitant ATP hydrolysis, and the addition of DNA also enhanced ATP hydrolysis by protease La 2-fold, but only in the presence of casein. At much higher concentrations, DNA inhibited proteolysis as well as ATP cleavage. Thus, association ofthis enzyme with DNA may regulate the degradation of cell proteins in vivo.The product of the Ion gene (also called capR or deg) in Escherichia coli (1) has recently been shown to be the ATP-dependent protease (2, 3), protease La (2, 4-6). This novel enzyme is an ATPase (2-4) as well as a serine protease (7,8), and inhibition of ATP hydrolysis also prevents proteolysis (2-6). This coupling of protein and ATP hydrolysis can account for the energy requirement for protein breakdown in intact cells (9-14). Furthermore, in lon-strains, the rates of degradation of abnormal proteins (14-17) and certain normal proteins (17, 18) are decreased. Thus, this protease appears to catalyze the rate-limiting steps in degradation of cell proteins in vivo (2,4,14).One additional important property of the Ion gene product is that it binds to DNA with high affinity (1). The precise biochemical reasons why these multiple functions (i.e., protein hydrolysis, ATP hydrolysis, and DNA binding) reside in the same protein are unclear. The present studies were undertaken to determine whether binding ofthe protease to DNA may alter its proteolytic function. One precedent for DNA affecting a proteolytic process is in the cleavage of bacteriophage A repressor and the lexA protein by the recA gene product (19,20). These specific cleavages which are essential for phage induction require the presence ofpolynucleotides and also ATP or an ATP analog (19,20). An influence of DNA binding on the activity of protease La could have profound physiological implications and may help explain why the ion gene product affects a wide variety of cellular processes (21), including the rapid degradation of certain DNA-binding proteins (18), the expression of certain genes (21), sensitivity to UV and other DNA-damaging agents (22, 23), and even cell division (24, 25). MATERIALS AND METHODSBacterial Strains. E. coli K-12 strain RGC121/pJMC40 was provided by A. Markovitz. This strain (1) carries the wild-type lon+ (capRI) allele on the chromosome and on the plasmid pSC101. These cells were grown in LB medium sup...

Functional mechanics of the ATP-dependent Lon protease- lessons from endogenous protein and synthetic peptide substrates

“…Different observations have been reported for the relationship between DNA binding and the enzymatic activities of bacterial Lon (51,53,54,56). One study showed that large DNA molecules such as plasmid, bacteriophage and calf thymus DNA stimulate the ATPase and protease activities of Lon (51). By contrast, another study observed that DNA stimulated ATP hydrolysis but inhibited proteolysis (53).…”

“…The SSD sub-region of the ATPase domain of B. thermoruber Lon is sufficient for DNA binding (55), whereas the ATPase domain lacking the SSD of EcLon also binds DNA (54). Different observations have been reported for the relationship between DNA binding and the enzymatic activities of bacterial Lon (51,53,54,56). One study showed that large DNA molecules such as plasmid, bacteriophage and calf thymus DNA stimulate the ATPase and protease activities of Lon (51).…”

“…EcLon binds to double-stranded DNA (dsDNA) with a relative higher affinity than to single-stranded DNA (ssDNA) (51,52). Several in vitro studies show that bacterial Lon interacts with large DNA molecules (51,53,54), whereas one study demonstrates sequencespecific EcLon binding to a 35 bp element referred to as pets (52). However, in a study conducted by a different group, the 35 bp pets element does not compete with the non-specific DNA binding of EcLon even at an 100-fold molar excess (54).…”

A Polyphosphate-Lon Protease Complex in the Adaptation ofEscherichia colito Amino Acid Starvation

“…16,17) Lon and two other proteases (ClpAP and ClpXP) are responsible for 70% to 80% of the energy-dependent protein degradation in E. coli. 5) We found that mutations in both the Lon and Clp proteases produce the same phenotype as ppk mutations and that the addition of amino acids overcomes the block in these cases.…”

Unusual observations during construction of a new cloning vector providing lon gene expression in Escherichia coli

“…Some results show that addition of DNA enhances ATP hydrolysis and that protease La apparently binds to DNA. It is possible that the binding of La to DNA has a significant influence on regulation of degradation of proteins (Chung and Goldberg, 1982).…”