Translation arrest of SecM is essential for the basal and regulated expression of SecA

Abstract

The SecM protein of Escherichia coli contains an arrest sequence (F 150 XXXXWIXXXXGIRAGP 166 ), which interacts with the ribosomal exit tunnel to halt translation elongation beyond Pro-166. This inhibition is reversed by active export of the nascent SecM chain. Here, we studied the physiological roles of SecM. Arrest-alleviating mutations in the arrest sequence reduced the expression of secA, a downstream gene on the same mRNA. Among such mutations, the arrest-abolishing P166A substitution mutation on the chromosomal secM gene proved lethal unless the mutant cells are complemented with excess SecA. Whereas secretion defect due either to azide addition, a secY mutation, or low temperature leads to up-regulated SecA biosynthesis, this regulation was lost by a secM mutation, which synergistically retarded growth of cells with lowered secretion activity. Finally, an arrest-alleviating rRNA mutation affecting the constricted part of the exit tunnel lowered the basal level of SecA as well as its secretion defect-induced upregulation. Thus, the arrest sequence of SecM has at least two roles in SecA translation. First, the transient elongation arrest in normal cells is required for the synthesis of SecA at levels sufficient to support cell growth. Second, the prolonged SecM elongation arrest under conditions of unfavorable protein secretion is required for the enhanced expression of SecA to cope with such conditions.

Nascent chain-monitored remodeling of the Sec machinery for salinity adaptation of marine bacteria

“…They up-regulate translation of secA and yidC2, respectively, in response to defects in the respective protein localization pathways, thereby contributing to the homeostasis of cellular protein localization processes (20)(21)(22)(23). SecM and MifM, called regulatory nascent polypeptides, undergo translation arrest in a regulated manner such that the arrest is released upon engagement of their nascent chains in the localization processes (17)(18)(19)(24)(25)(26).…”

Recruitment of a species-specific translational arrest module to monitor different cellular processes

“…We therefore added a myc-tag to the C terminus of MifM to extend the full-length product to an extent separable from the arrested polypeptide moiety by SDS/PAGE. When mifM-myc was translated with Bs hybrid PURE system, the 30 kDa band again appeared earlier (at 5 min; lane 12) than the other major band, which now migrated at the position of 12 kDa and became detectable at 10 min (lanes [13][14][15]. This 12 kDa band must represent the full-length MifM-myc polypeptide because it is resistant to RNaseA (lanes [18][19][20] and uniquely observed after the myc-tag addition (compare lanes 2-5 and lanes 12-15).…”

“…The stalled ribosome uncovers the SD sequence required for translation of secA (12). Therefore, the elongation arrest, which is transient in normal cells but prolonged in secretion-compromised cells, assures the basal level expression of secA as well as its up-regulation in response to a secretion defect (13). MifM monitors YidC-dependent membrane biogenesis pathway in B. subtilis, which has two YidC homologs, the primary SpoIIIJ (YidC1) and the secondary YidC2 (5,14).…”

The Long α-Helix of SecA Is Important for the ATPase Coupling of Translocation

“…Plasmid pKY173 was an SecA overproducer (29), whereas pNA83 was its derivative encoding cysteine-less SecA. pAN5 was another SecA overproducer, which was constructed on a vector compatible with pKY173 (28). Sitedirected mutations were introduced by the QuikChange (Stratagene) method.…”