Backbone and side chain NMR assignment, along with the secondary structure prediction of RRM2 domain of La protein from a lower eukaryote exhibiting identical structural organization with its human homolog

Abstract

The La protein (Lupus antigen), a key mediator during biogenesis of RNA polymerase III transcripts, contains a characteristic La motif and one or two RNA recognition motif (RRM) domains, depending on the organism of origin. The RRM1 domain is conserved in higher eukaryotes and located in the N-terminal region, whereas the C-terminal RRM2 domain is absent in most lower eukaryotes and its specific role remains, so far, uncharacterized. Here, we present the backbone and side-chain assignment of the (1)H, (13)C and (15)N resonances of RRM2 of La protein from Dictyostelium discoideum. Interestingly, the La protein in this lower eukaryote, exhibits high homology to its human counterpart. Moreover, it contains two RRM domains, instead of one, raising questions on its evolutionary origin and the putative role of RRM2 in vivo. We also provide its secondary structure as predicted by the TALOS+ online tool.

Quantifying side-chain conformational variations in protein structure

“…Nevertheless, the side-chain variations have not yet been quantitatively analyzed, the understanding of side-chain conformational variation is still intuitive and not systematic. Side-chain conformation prediction, or side-chain packing, has been a well-established problem in computational biology and involved in diverse applications, such as protein folding3, docking4, design5, engineering6 and structure optimization7. Various new programs89101112, which do not consider conformational change, have been proposed in recent years.…”

Studies on the role of aminoacyl-tRNA synthesis in the regulation of ribosomal and exo-ribosomal protein synthesis in pathogens

“…Both biochemical and structural studies from our group and other group have shown that La protein exhibits conserved functional domains which contribute to the overall recognition of its substrates (except the 3'OH-UUU termini) and facilitate their proper folding and maturation. Such individual domains are the well characterized La motif which seems to exhibit the recognition specificity and one or more RRM motifs (RNA binding motifs) whose actual role is under investigation (Figure 5) [30,31,32].…”

“…Each 5' *γ- 32 P]-tRNA Gly transcript (up to 0.1 μΜ in the presence of unlabeled tRNA Gly transcript) was mixed at the same time with each glyS T-box variant (5 μΜ) in 1x binding buffer 15 and was denatured for 3 min at 65 o C. After refolding at RT samples were incubated at 25 o C for 1 to 1.5 h. All reactions were performed in a final volume of 10 μL and were stopped with glycerol (8% v/v). Moreover, reaction mixtures without glyS T-box were used as a negative control.…”

“…3.3., without the dephosphorylation step. In primer extension reactions 2 pmol of each modified transcript were hybridized with 5' *γ-32 P]labeled TbGl_1 or TbGl_6 primer in 1x hybridization buffer22 (final volume 10 μL). Reactions were incubated for 10 min at 80 o C, followed by slowly cooling in water bath to 47 o C. Subsequently, extension reactions were induced by addition of 1x primer extension buffer 23(23 μL) in presence of 2U RNasin and 5U AMV reverse transcriptase in a final volume of 42 μL and were incubated at 47 o C for 1 h. After addition of 158 μL 1x TE buffer, the prodused cDNAs were ethanol precipitated, resuspented in 4 μL of 0.1 N NaOH, and 8 μL of f-EDTA loading buffer24 , and were denatured at 80 o C for 2 min, prior being analyzed on 6% PAGE/Urea.…”

“…Precipitation derivates were resuspendent in H 2 O (DEPC treated) up to 1 pmol/μL. Subsequently, 2.5 to 5 pmol of dephosphorylated transcripts were phosporylated at the 5' end with *γ-32 P]-ATP by T4 polynucleotide kinase reaction: Running the prediction tool to the automated mode (http://swissmodel.expasy.org/workspace), the protein can be modeled in 3D, according to its amino acid chain organization by similarity matching with another protein sequence, whose tertiary structure has already been experimentally verified and annotated. T. thermophilus GlyRS (Uniprot No: P56206; PDB ID: 1ati)[259] represents a Class II (α 2 ) homolog with characterized structural features and it was chosen for the homology modeling procedure, possessing 41 % identity in sequence with the S. epidermidis protein.…”