SRP19 Is a Dispensable Component of the Signal Recognition Particle in Archaea

Abstract

In vitro, archaeal SRP54 binds SRP RNA in the absence of SRP19, suggesting the latter to be expendable in Archaea. Accordingly, the Haloferax volcanii SRP19 gene was deleted. Although normally transcribed at a level comparable to that of the essential SRP54 gene, SRP19 deletion had no effect on cell growth, membrane protein insertion, protein secretion, or ribosome levels. The absence of SRP19 did, however, increase membrane bacterioruberin levels.Despite their structural similarities (9, 28), eukaryal and archaeal signal recognition particles (SRP) seemingly follow very different pathways of assembly. In Eukarya, SRP19 binding both to the capping tetraloop of SRP RNA helix 6 and to a lower-affinity binding site near the tip of helix 8 brings these helices into close proximity, leading to a conformational change in helix 8 and exposure of the normally cryptic SRP54-binding site (16,21). By contrast, structural and biochemical investigations of archaeal SRP raise questions regarding the role of SRP19 in the assembly of the particle in Archaea. While crystallographic studies have shown the SRP54 binding site to be fully presented in the SRP19-SRP RNA complex (11), structural analysis of free archaeal SRP RNA has shown that helices 6 and 8 normally lie parallel to each other and interact (12,27) and that there is partial exposure of the SRP RNA helix 8 SRP54-binding site (12). Accordingly, in vitro reconstitutions with purified components from Archaeoglobus fulgidus (4, 7), Methanococcus jannaschii (12), Pyrococcus furiosus (18), or Haloferax volcanii (26) have shown binding of archaeal SRP54 to SRP RNA in the absence of SRP19. Although these studies showed that SRP19 was required for high-affinity binding, SRP54 clearly presented inherent affinity for SRP RNA (7, 12). Thus, although polyhistidine-tagged SRP19, SRP54, and SRP RNA could be cocaptured from transformed H. volcanii cells (22), the need for SRP19 in vivo merits further investigation.To discern whether SRP19 is essential in H. volcanii, the encoding gene was exchanged for the tryptophan synthaseencoding trpA gene by a gene knockout technique developed for H. volcanii (2, 5). Briefly, a pyrE-containing vector plasmid encoding 400 nucleotides flanking each end of the SRP19 gene, separated by trpA, was integrated into the genome of H. volcanii strain WR536, a uracil and tryptophan auxotroph. To replace SRP19, the transformed cells were grown in the presence of uracil and 5-fluoroorotic acid, without tryptophan. PCR using primers directed against the SRP19 flanking regions, designed to follow the exchange of trpA for SRP19, revealed plasmid integration, with bands corresponding to genome-and plasmid-derived sequences being detected in the transformed cells (Fig. 1A, lane 2). Upon expulsion of SRP19 and the integrated plasmid, only trpA with its SRP19 flanking regions were detected (lane 3). Using genomic DNA from untreated and SRP19-lacking cells as a template and primers directed against the SRP19 coding region, PCR revealed a reaction product in the backgr...

aglF, aglG and aglI, novel members of a gene island involved in the N‐glycosylation of the Haloferax volcanii S‐layer glycoprotein

“…Real‐time RT‐PCR was performed essentially as described (Yurist et al ., 2007) using SYBR Green PCR 2× Master Mix (Applied Biosystems, Foster City, CA). Primers (listed in Table 2) were designed using Primer Express 2.0 software (PerkinElmer Life Sciences) and employed at a final concentration of 400 nM.…”

Mutations in signal recognition particle SRP54 cause syndromic neutropenia with Shwachman-Diamond–like features

“…These proteins have a de facto need for SRP54 for their synthesis. Although homozygous mutathe exception of the SRP RNA, SRP54 is the only one of the six SRP polypeptides that is universally conserved throughout evolution (31,32), with 96% sequence identity between human and zebrafish molecules. This high level of conservation is explained by the central role played by SRP54 in the cotranslational process that affects over one-third of the human proteome (see below).…”

Protein transport across and into cell membranes in bacteria and archaea

“…In Eukarya, SRP19 is involved in SRP assembly and facilitates the binding of SRP54 to the SRP RNA by interacting with SRP RNA helix 8 [67][68][69][70]. Nevertheless, it has been shown that, unlike in Eukarya, archaeal SRP19 is not strictly required for the binding of archaeal SRP RNA to SRP54/Ffh [67,71,72]. The SRP19-independent binding of archaeal SRP RNA and SRP54/Ffh seems to reflect the stability of the SRP complex, which may be required for growth at extreme temperatures or pH values or in highly saline environments [68].…”