Conformation of 4.5S RNA in the signal recognition particle and on the 30S ribosomal subunit.
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 wild-type 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' 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.
Gu
Shan-Qing
SQ
Institute of Molecular Biology, University of Witten/Herdecke, Stockumer Str. 10, 58448 Witten, Germany.
Jöckel
Johannes
J
Beinker
Philipp
P
Warnecke
Jens
J
Semenkov
Yuri P
YP
Rodnina
Marina V
MV
Wintermeyer
Wolfgang
W
D001483:Base Sequence; D002151:Calorimetry; D004926:Escherichia coli / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism*; D012329:RNA, Bacterial; D012335:RNA, Ribosomal / Q000737:chemistry / Q000378:metabolism*; D012343:RNA, Transfer / Q000378:metabolism; D012269:Ribosomal Proteins / Q000737:chemistry / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*; D018271:Signal Recognition Particle / Q000378:metabolism*
C409968:4.5S RNA; D029968:Escherichia coli Proteins; C082931:Ffh protein, E coli; D021122:Protein Subunits; D012329:RNA, Bacterial; D012335:RNA, Ribosomal; D012269:Ribosomal Proteins; D018271:Signal Recognition Particle; D012343:RNA, Transfer