Crystallization and preliminary X-ray analysis of the conserved domain IV of Escherichia coli 4.5S RNA. Erratum (original) (raw)
Related papers
Conformation of 4.5S RNA in the signal recognition particle and on the 30S ribosomal subunit
RNA, 2005
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. .
RNA
The structure of 4.5S RNA, the Escherichia coli homologue of the signal recognition particle (SRP) RNA, alone and in the SRP complex with protein P48 (Ffh) was probed both enzymatically and chemically. The molecule is largely resistant against single strand-specific nucleases, indicating a highly base paired structure. Reactivity appears mainly in the apical tetraloop and in one of the conserved internal loops. Although some residues are found reactive toward dimethylsulphate and kethoxal in regions predicted to be unpaired by the phylogenetic secondary structure model of 4.5S RNA, generally the reactivity is low, and some residues in internal loops are not reactive at all. RNase V1 cleaves the RNA at multiple sites that coincide with predicted helices, although the cleavages show a pronounced asymmetry. The binding of protein P48 to 4.5S RNA results in a protection of residues in the apical part of the molecule homologous to eukaryotic SRP RNA (domain IV), whereas the cleavages in ...
Formation of SRP-like particle induces a conformational change in E. coli 4.5S RNA
FEBS Letters, 1994
E. coli P48 protein is homologous to the SRP54 component of the eukaryotic signal recognition particle. In vivo, P48 is associated with 4.5s RNA which shares a homology with eukaryotic SRP RNA. To study the interaction between P48 and 4.5s RNA in vitro, we used 4.5s RNA with fluorescein coupled to the 3'-terminal ribose. Upon binding of P48, the fluorescent 4.5s RNA shows a substantial decrease in fluorescence. Fluorescence quenching as well as anisotropy measurements reveal that the effect is not due to a direct interaction of P48 with the dye. This suggests that the binding of P48 induces a conformational change in 4% RNA which affects the structure at the 3' end of the RNA. From equilibrium titrations with fluorescent 4.5s RNA, a dissociation constant of 0.15 pm is obtained for the RNA. protein complex. The formation of the complex is not affected by GTP binding to or hydrolysis by P48.
Structure, 2000
Background: Bacterial signal recognition particle (SRP), consisting of 4.5S RNA and Ffh protein, plays an essential role in targeting signal-peptidecontaining proteins to the secretory apparatus in the cell membrane. The 4.5S RNA increases the affinity of Ffh for signal peptides and is essential for the interaction between SRP and its receptor, protein FtsY. The 4.5S RNA also interacts with elongation factor G (EF-G) in the ribosome and this interaction is required for efficient translation.
Inter-RNA homology and possible roles of small RNAs
Journal of Molecular Evolution, 1981
The nucleotide sequence of a segment of U1 and U3b small RNAs (sRNAs) is shown to have a high complementarity with the nucleotide sequence of a part of the leader region of almost all eukaryotic genes studied so far. The complementary region of U3b is located in the unpaired segment of the secondary structure of U3b constructed by . A similar complementarity is also observed between these RNAs and the leader regions of eukaryotic viruses, but the complementary region is not always identical with that for eukaryotic genes. Complementarity is also observed between the 3' end of 18S rRNA and a segment of U1 or U3b which is almost contiguous to the region complementary with mRNA. These observations suggest that U1 and U3b may be involved in mRNA processing and transport in the nucleus or in translation in the cytoplasm. In addition to U1 and U3b, another sRNA, i.e., 4.5S RNA I, is shown to have segments which are homologous to the Hogness box of the flanking region of gene and the Proudfoot-Brownlee (PB) box of mRNA near the poly(A) attachment site. The two segments which are complementary with these boxes are located almost contiguously on a co-joined loop of the secondary structure of 4.5S RNA I constructed by . Since the Hogness box and PB box are both considered as a recognition site by the RNA polymerase, it is possible that 4.5S RNA I is involved in mediating gene transcription.
RNA, 2008
The signal recognition particle (SRP) plays a pivotal role in transporting proteins to cell membranes. In higher eukaryotes, SRP consists of an RNA molecule and six proteins. The largest of the SRP proteins, SRP72, was found previously to bind to the SRP RNA. A fragment of human SRP72 (72c9) bound effectively to human SRP RNA but only weakly to the similar SRP RNA of the archaeon Methanococcus jannaschii. Chimeras between the human and M. jannaschii SRP RNAs were constructed and used as substrates for 72c9. SRP RNA helical section 5e contained the 72c9 binding site. Systematic alteration within 5e revealed that the A240G and A240C changes dramatically reduced the binding of 72c9. Human SRP RNA with a single A240G change was unable to form a complex with full-length human SRP72. Two small RNA fragments, one composed of helical section 5ef, the other of section 5e, competed equally well for the binding of 72c9, demonstrating that no other regions of the SRPR RNA were required. The biochemical data completely agreed with the nucleotide conservation pattern observed across the phylogenetic spectrum. Thus, most eukaryotic SRP RNAs are likely to require for function an adenosine within their 5e motifs. The human 5ef RNA was remarkably resistant to ribonucleolytic attack suggesting that the 240-AUC-242 ''loop'' and its surrounding nucleotides form a peculiar compact structure recognized only by SRP72. .