Identification and Sequence Analysis of Contact Sites between Ribosomal Proteins and rRNA in Escherichia coli 30 S Subunits by a New Approach Using Matrix-assisted Laser Desorption/Ionization-Mass Spectrometry Combined with N-terminal Microsequencing

Abstract

The quarternary structure of the ribosome from the eubacterium Escherichia coli has been studied intensively for many years, and several individual models concerning either the protein composition and the arrangement of proteins (e.g. Refs. 1-5) or the three-dimensional folding of 16 S RNA including the protein-rRNA interactions (e.g. Refs. 6 -9) have been proposed. However, the resolution of these models at the molecular level is still limited, since the contact sites between adjacent proteins and between proteins and rRNA are not precisely known at the amino acid level with only a few exceptions of crosslinked protein pairs (e.g. Refs. 10 -12), although a number of protein contact sites have been identified at the nucleotide level (for review, see Ref. 9). More recently, detailed protein-rRNA cross-linking studies at the peptide level (13) in combination with three-dimensional structures of isolated ribosomal proteins (e.g. Refs. 14 -17) have provided more insight into the protein-RNA interactions and their functional implications within the prokaryotic ribosome. Additionally, site-directed hydroxyl radical probing of the rRNA neighborhood in reconstituted ribosomal particles has provided information relevant to the three-dimensional orientation of several proteins within the 30 S subunit (18,19).Even though discrete peptide regions of individual ribosomal proteins in close contact to the rRNA have been clearly established (13), the analysis of the corresponding sites on the rRNA has still remained a problem. Detailed modeling of ribosomal structures requires precise knowledge of protein-nucleotide and peptide-RNA contact sites concomitantly at the amino acid and nucleotide level. With the help of such information, the three-dimensional structures of ribosomal proteins can be placed into current models of the rRNA, and hence refined models can be adapted to the overall topography of the prokaryotic ribosome as derived from cryo-electron microscopy data (20,21). For this purpose we have developed a new approach that enables us to determine simultaneously the contact sites at the peptide as well as at the nucleotide level within cross-linked peptide-oligoribonucleotide complexes isolated from ribosomal subunits. The strategy employed is based on N-terminal sequence analysis combined with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). 1 MALDI-MS (22) has been successfully applied for e.g. the determination of phosphoproteins, glycoproteins, and oligonucleotides (for review, see . Furthermore, applications of MALDI-MS for sequencing of peptides (e.g. Refs. 26 -28) and of oligodeoxynucleotides (e.g. Refs. 29 and 30) have been described. Here, we report for the first time the sequencing of oligoribonucleotides by MALDI-MS cross-linked to peptides. The strategy applied here should be a valuable tool for studying protein-RNA interactions, not only in ribosomes but also in other protein⅐RNA complexes. EXPERIMENTAL PROCEDURESPreparation of 30 S ribosomal subunits from E. coli (Eco 30S), chemic...

Ribosomal Protein S14 of Saccharomyces cerevisiae Regulates Its Expression by Binding to RPS14B Pre-mRNA and to 18S rRNA

“…The conformation of rRNA in ribosomes containing a streptomycin-resistant or -dependent mutant S12 protein is altered compared to that of rRNA in wild-type cells (1). As mentioned previously, antibiotic resistance mutations in several r proteins are located in or near amino acids that can be cross-linked to rRNA (8,10,18,49,51,60,61). The observation that drug resistance mutations in rpS14 alter the interaction of this protein with its two RNA targets demonstrates that resistance mutations reside in RNA binding domains of r proteins.…”

“…Like many ribosomal proteins, the amino acid sequence of rpS14 does not contain a discernible RNA recognition domain. However, analyses of bacterial r proteins suggest that conserved, basic amino acids, particularly those located in loops or turns in the protein structure, conserved solvent-exposed hydrophobic residues, and amino acids mutable to drug resistance phenotypes are hallmarks of RNA binding domains in r proteins (60,61; reviewed in references 49 and 50). These observations suggest that the C terminus of rpS14 might be involved in RNA recognition because it is rich in highly conserved, basic residues (30) that are predicted (by the Chou-Fasman algorithm) to fold into a loop-turn structure.…”

Merging Molecular Electron Microscopy and Mass Spectrometry by Carbon Film-assisted Endoproteinase Digestion

“…7B). Of note, RNases are surprisingly stable against endoproteolytic digestion (34,35). However, the RNases were not present in the elution profile of the ECAD samples (Fig.…”

Nested Arg-specific bifunctional crosslinkers for MS-based structural analysis of proteins and protein assemblies

“…The broad applicability of soft ionization techniques, such as matrix assisted laser desorption ionization (MALDI) [10,11] and electrospray ionization (ESI) [12,13], and the ability to perform sequence determinations [14,15] make mass spectrometry (MS) the analytical platform of choice for the characterization of products obtained from probing reactions. For this reason, approaches based on crosslinking and MS detection have been successfully applied to individual substrates of proteic [16][17][18][19][20][21][22][23][24] and nucleic acid nature [25,26] and to assemblies of increased complexity involving specific protein-peptide [27,28], protein-protein [29][30][31][32][33][34][35][36][37][38][39][40][41], and protein-nucleic acid [42][43][44][45][46][47][48][49][50][51][52][53][54] interactions.…”