Abstract:L7Ae is a member of a protein family that binds kink-turns (k-turns) in many functional RNA species. We have solved the X-ray crystal structure of the near-consensus sequence Kt-7 of Haloarcula marismortui bound by Archaeoglobus fulgidus L7Ae at 2.3-Å resolution. We also present a structure of Kt-7 in the absence of bound protein at 2.2-Å resolution. As a result, we can describe a general mode of recognition of k-turn structure by the L7Ae family proteins. The protein makes interactions in the widened major gr… Show more
“…These residues make close van der Waals contacts with the L1 and L2 nts of the K-turn [for examples, see (28,32)], with E94, the only charged amino acid in this loop, forming a more specific interaction through hydrogen bonding to L1 (32). V95 also forms one side of the hydrophobic pocket that binds the extruded L3 nt (30,31).…”
The RNA-binding protein L7Ae, known for its role in translation (as part of ribosomes) and RNA modification (as part of sn/oRNPs), has also been identified as a subunit of archaeal RNase P, a ribonucleoprotein complex that employs an RNA catalyst for the Mg2+-dependent 5′ maturation of tRNAs. To better understand the assembly and catalysis of archaeal RNase P, we used a site-specific hydroxyl radical-mediated footprinting strategy to pinpoint the binding sites of Pyrococcus furiosus (Pfu) L7Ae on its cognate RNase P RNA (RPR). L7Ae derivatives with single-Cys substitutions at residues in the predicted RNA-binding interface (K42C/C71V, R46C/C71V, V95C/C71V) were modified with an iron complex of EDTA-2-aminoethyl 2-pyridyl disulfide. Upon addition of hydrogen peroxide and ascorbate, these L7Ae-tethered nucleases were expected to cleave the RPR at nucleotides proximal to the EDTA-Fe–modified residues. Indeed, footprinting experiments with an enzyme assembled with the Pfu RPR and five protein cofactors (POP5, RPP21, RPP29, RPP30 and L7Ae–EDTA-Fe) revealed specific RNA cleavages, localizing the binding sites of L7Ae to the RPR's catalytic and specificity domains. These results support the presence of two kink-turns, the structural motifs recognized by L7Ae, in distinct functional domains of the RPR and suggest testable mechanisms by which L7Ae contributes to RNase P catalysis.
“…These residues make close van der Waals contacts with the L1 and L2 nts of the K-turn [for examples, see (28,32)], with E94, the only charged amino acid in this loop, forming a more specific interaction through hydrogen bonding to L1 (32). V95 also forms one side of the hydrophobic pocket that binds the extruded L3 nt (30,31).…”
The RNA-binding protein L7Ae, known for its role in translation (as part of ribosomes) and RNA modification (as part of sn/oRNPs), has also been identified as a subunit of archaeal RNase P, a ribonucleoprotein complex that employs an RNA catalyst for the Mg2+-dependent 5′ maturation of tRNAs. To better understand the assembly and catalysis of archaeal RNase P, we used a site-specific hydroxyl radical-mediated footprinting strategy to pinpoint the binding sites of Pyrococcus furiosus (Pfu) L7Ae on its cognate RNase P RNA (RPR). L7Ae derivatives with single-Cys substitutions at residues in the predicted RNA-binding interface (K42C/C71V, R46C/C71V, V95C/C71V) were modified with an iron complex of EDTA-2-aminoethyl 2-pyridyl disulfide. Upon addition of hydrogen peroxide and ascorbate, these L7Ae-tethered nucleases were expected to cleave the RPR at nucleotides proximal to the EDTA-Fe–modified residues. Indeed, footprinting experiments with an enzyme assembled with the Pfu RPR and five protein cofactors (POP5, RPP21, RPP29, RPP30 and L7Ae–EDTA-Fe) revealed specific RNA cleavages, localizing the binding sites of L7Ae to the RPR's catalytic and specificity domains. These results support the presence of two kink-turns, the structural motifs recognized by L7Ae, in distinct functional domains of the RPR and suggest testable mechanisms by which L7Ae contributes to RNase P catalysis.
“…The 15.5k protein fails to bind to the k-turn when the A1n is methylated at the exocyclic N6. All the specific interactions between this class of protein and k-turns are made with the conserved guanine nucleotides at the 1b and 2n positions that lie in the major groove on the outside of the kinked RNA, whereas the A1n is on the inside of the EMBO reports N 6 -methyladenine blocks snoRNA assembly Lin Huang et al structure making no contact with the protein [35]. This class of protein therefore makes indirect recognition of the methylation, resulting from the overall RNA structure rather than specific contacts.…”
Section: Humanmentioning
confidence: 99%
“…L7Ae protein Archeoglobus fulgidus L7Ae protein was expressed and purified as previously published [35]. protein eluted at 500 mM NaCl.…”
Section: Preparation Of Proteinsmentioning
confidence: 99%
“…Binding of L7Ae-family proteins induces the formation of the tightly kinked k-turn structure [33][34][35] (Fig 1B). The core of the k-turn structures formed by the box C/D and C'/D' elements comprises consecutive trans sugar-Hoogsteen GÁA base pairs (sometimes termed sheared base pairs) that position the conserved adenine nucleobases to make key cross-strand hydrogen bonds that stabilize the conformation ( Fig 1C).…”
N6‐methyladenine is the most widespread mRNA modification. A subset of human box C/D snoRNA species have target GAC sequences that lead to formation of N6‐methyladenine at a key trans Hoogsteen‐sugar A·G base pair, of which half are methylated in vivo. The GAC target is conserved only in those that are methylated. Methylation prevents binding of the 15.5‐kDa protein and the induced folding of the RNA. Thus, the assembly of the box C/D snoRNP could in principle be regulated by RNA methylation at its critical first stage. Crystallography reveals that N6‐methylation of adenine prevents the formation of trans Hoogsteen‐sugar A·G base pairs, explaining why the box C/D RNA cannot adopt its kinked conformation. More generally, our data indicate that sheared A·G base pairs (but not Watson–Crick base pairs) are more susceptible to disruption by N6mA methylation and are therefore possible regulatory sites. The human signal recognition particle RNA and many related Alu retrotransposon RNA species are also methylated at N6 of an adenine that forms a sheared base pair with guanine and mediates a key tertiary interaction.
“…Significantly, we have recently shown that Kt-7 can adopt either an N3 or N1 structure depending on its environment. While it adopts the N1 structure in the ribosome, it forms an N3 structure when replacing the natural k-turn of the SAM-I riboswitch (21), or as a simple duplex RNA either free or bound to the L7Ae protein (22). Figure 1.k-turn sequence and a key A-minor interaction.…”
Kt-23 from Thelohania solenopsae is a rare RNA kink turn (k-turn) where an adenine replaces the normal guanine at the 2n position. L7Ae is a member of a strongly conserved family of proteins that bind a range of k-turn structures in the ribosome, box C/D and H/ACA small nucleolar RNAs and U4 small nuclear RNA. We have solved the crystal structure of T. solenopsae Kt-23 RNA bound to Archeoglobus fulgidus L7Ae protein at a resolution of 2.95 Å. The protein binds in the major groove displayed on the outer face of the k-turn, in a manner similar to complexes with standard k-turn structures. The k-turn adopts a standard N3 class conformation, with a single hydrogen bond from A2b N6 to A2n N3. This contrasts with the structure of the same sequence located in the SAM-I riboswitch, where it adopts an N1 structure, showing the inherent plasticity of k-turn structure. This potentially can affect any tertiary interactions in which the RNA participates.
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