Conformation of 4.5S RNA in the signal recognition particle and on the 30S ribosomal subunit

Abstract

The signal recognition particle (SRP) from Escherichia coli consists of 4.5S RNA and protein Ffh. It is essential for targeting ribosomes that are translating integral membrane proteins to the translocation pore in the plasma membrane. Independently of Ffh, 4.5S RNA also interacts with elongation factor G (EF-G) and the 30S ribosomal subunit. Here we use a cross-linking approach to probe the conformation of 4.5S RNA in SRP and in the complex with the 30S ribosomal subunit and to map the binding site. The UV-activatable cross-linker p-azidophenacyl bromide (AzP) was attached to positions 1, 21, and 54 of wildtype or modified 4.5S RNA. In SRP, cross-links to Ffh were formed from AzP in all three positions in 4.5S RNA, indicating a strongly bent conformation in which the 5 0 end (position 1) and the tetraloop region (including position 54) of the molecule are close to one another and to Ffh. In ribosomal complexes of 4.5S RNA, AzP in both positions 1 and 54 formed cross-links to the 30S ribosomal subunit, independently of the presence of Ffh. The major cross-linking target on the ribosome was protein S7; minor cross-links were formed to S2, S18, and S21. There were no cross-links from 4.5S RNA to the 50S subunit, where the primary binding site of SRP is located close to the peptide exit. The functional role of 4.5S RNA binding to the 30S subunit is unclear, as the RNA had no effect on translation or tRNA translocation on the ribosome.

Interaction of signal-recognition particle 54 GTPase domain and signal-recognition particle RNA in the free signal-recognition particle

“…A direct interaction between the NG domain and the SRP RNA in free SRP has been suggested by prior biochemical studies (17,18,(24)(25)(26). The NG-RNA interactions observed in S domain A are consistent with some but not all of these data.…”

“…These show different relative orientations of the NG domain at two different stages in the SRP cycle. In both the mammalian and S. solfataricus structures, SRP54 assumes a so-called ''open'' conformation, in which the NG domain and the RNA are separated by at least 20 Å. Biochemical data, however, indicate that, in free SRP, the NG domain can directly interact with SRP RNA (17,18,(24)(25)(26). To further address this discrepancy and to better delineate the structural basis of the first step in the SRP cycle, we have determined the crystal structure of the free S domain of SRP of the Archaeon Methanococcus jannaschii at 2.5-Å resolution.…”

SRP RNA provides the physiologically essential GTPase activation function in cotranslational protein targeting

“…Nonetheless, these results do not address the role of the 4.5S RNA, which also stimulates GTPase activity in the SRP-FtsY complex in vitro and is essential for SRP function in vivo (Brown and Fournier 1984;Peluso et al 2001;Sagar et al 2004;Gu et al 2005). A truncated form of the 114-nucleotide (nt) RNA, including just the distal 44 nt at the hairpin end (domain IV) of the molecule, is sufficient to support cell growth ( Fig.…”

Kinship in the SRP RNA family

“…The SRP54/ Ffh-RNA complex is a target for intense investigations in attempts to explain its role in signal peptide binding and release. [51][52][53][54][55][56] It has been suggested that helix 8 may interact directly with the N-terminal portion of the signal peptide. 39 Recently, evidence has been provided that this region of the SRP RNA releases the autoinhibition of the mutually controlled GTPase activities between the signal-bound SRP and the SRP receptor (SR).…”