Protein-rRNA binding features and their structural and functional implications in ribosomes as determined by cross-linking studies.

Urlaub, Henning; Kruft, Volker; Bischof, Oliver F.; Müller, Eva; Wittmann‐Liebold, Brigitte

doi:10.1002/j.1460-2075.1995.tb00137.x

Cited by 112 publications

(113 citation statements)

References 66 publications

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“…However, a crosslink between L6 and the loop of helix 89 around nucleotide 2475 (31,42) is not covered by the L6 binding site reported here (see arrow in Fig. 5A).…”

Section: Resultsmentioning

confidence: 60%

Selecting rRNA binding sites for the ribosomal proteins L4 and L6 from randomly fragmented rRNA: Application of a method called SERF

Stelzl

Spahn

Nierhaus

2000

Proc. Natl. Acad. Sci. U.S.A.

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Two-thirds of the 54 proteins of the Escherichia coli ribosome interact directly with the rRNAs, but the rRNA binding sites of only a very few proteins are known. We present a method (selection of random RNA fragments; SERF) that can identify the minimal binding region for proteins within ribonucleo-protein complexes such as the ribosome. The power of the method is exemplified with the ribosomal proteins L4 and L6. Binding sequences are identified for both proteins and characterized by phosphorothioate footprinting. Surprisingly, the binding region of L4, a 53-nt rRNA fragment of domain I of 23S rRNA, can simultaneously and independently bind L24, one of the two assembly initiator proteins of the large subunit. Identifying the minimal length of RNA sequences that bind specifically to a protein is a challenge for structural research. The classical approach of digesting an RNA-protein complex with RNase usually does not give the minimal binding region, because the protein(s) within the complex might hinder the access of an RNase. Alternatively, the RNase can cut the RNA within the complex into short sequences that lose the binding capacity. Crosslinking approaches and protection experiments with base modifying reagents indicate vicinity but not necessarily the binding sequence. We decided to exploit the enormous power of in vitro selection methods that can identify the best fitting RNA sequences from about 10 15 variants (SELEX, systematic evolution of ligands by exponential enrichment; ref. 1). However, when randomized sequences are used as starting conditions, affinity selection methods usually do not lead to the naturally occurring binding site for a target as such.Short random fragments from genomic DNA were used to select the DNA binding site of transcription factor IIIA in vitro (2), and a similar idea has been put forward for the selection of protein-nucleic acid interactions (3). In vivo small DNA fragments randomly obtained from 14 genes were identified that stimulate expression of the lacZ gene (4). Expression libraries made of short rRNA sequences have been used to create resistance against antibiotics in vivo (5, 6).The basic idea presented here is that the use of a pool of random fragments from large RNAs directly reveals the native binding site when selected for affinity to a certain protein in vitro. The SELEX variant is abbreviated SERF for selection of random RNA fragments. The principle is shown in Fig. 1. A pool of random rDNA fragments is generated by a random cutting of rDNA. The rDNA fragments are transcribed into RNA in a second step. The advantages of the SERF method are: (i) the selection does not work on just any RNA-protein interactions but rather on the native ones; (ii) the size of the selected rRNA can be chosen and a series of overlapping fragments can reveal the minimal binding site; and (iii) the selection should be fast and efficient because the variability of the pool is low compared with the starting pool used in classical affinity selection experiments.More than two-thirds of t...

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“…However, a crosslink between L6 and the loop of helix 89 around nucleotide 2475 (31,42) is not covered by the L6 binding site reported here (see arrow in Fig. 5A).…”

Section: Resultsmentioning

confidence: 60%

Selecting rRNA binding sites for the ribosomal proteins L4 and L6 from randomly fragmented rRNA: Application of a method called SERF

Stelzl

Spahn

Nierhaus

2000

Proc. Natl. Acad. Sci. U.S.A.

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“…Some of the more successful approaches for characterizing ribosomes include crosslinking [1][2][3][4], cryo-electron microscopy (cryo-EM) [5][6][7], tritium bombardment [8], nuclear magnetic resonance (NMR) [9] and electrospray mass spectrometry [10][11][12][13]. One of the most successful approaches has been the development of X-ray crystallography within the past several years, wherein high-resolution crystal structures of ribosomal subunits or intact ribosomes have been obtained that provide detailed structural information at the atomic level [14][15][16][17][18].…”

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confidence: 99%

Developing limited proteolysis and mass spectrometry for the characterization of ribosome topography

Suh

Pourshahian

Limbach

2007

J. Am. Soc. Mass Spectrom.

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An approach that combines limited proteolysis and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been developed to probe protease-accessible sites of ribosomal proteins from intact ribosomes. Escherichia coli and Thermus thermophilus 70S ribosomes were subjected to limited proteolysis using different proteases under strictly controlled conditions. Intact ribosomal proteins and large proteolytic peptides were recovered and directly analyzed by MALDI-MS, which allows for the determination of proteins that are resistant to proteolytic digestion by accurate measurement of molecular weights. Larger proteolytic peptides can be directly identified by the combination of measured mass, enzyme specificity and protein database searching. Sucrose density gradient centrifugation revealed that the majority of the 70S ribosome dissociates into intact 30S and 50S subunits after 120 min of limited proteolysis. Thus, examination of ribosome populations within the first 30-60 min of incubation provide insight into 70S structural features. Results from E. coli and T. thermophilus revealed that a significantly larger fraction of 50S ribosomal proteins have similar limited proteolysis behavior than the 30S ribosomal proteins of these two organisms. The data obtained by this approach correlate with information available from the high resolution crystal structures of both organisms. This new approach will be applicable to investigations of other large ribonucleoprotein complexes, is readily extendable to ribosomes from other organisms, and can facilitate additional structural studies on ribosome assembly intermediates.The ribosome, found in all organisms, is the subcellular organelle that performs the activity of protein synthesis. Prokaryotic ribosomes, which sediment at 70S, have a molecular mass of approximately 2.5 ×10 6 Da and consist of two subunits, the 50S and the 30S. Each subunit has a unique number of proteins and ribosomal RNAs (rRNAs). Eukaryotic ribosomes, which sediment at 80S, are substantially larger and more complex than their prokaryotic counterparts. Two subunits, the 60S and 40S, together contain at least 78 unique proteins and 4 rRNAs. The small subunit binds messenger RNA (mRNA) and mediates the interactions between mRNA and transfer RNAs (tRNAs). The larger subunit catalyzes peptide-bond formation. During the initiation phase of protein synthesis, the two subunits behave independently, assembling into complete ribosomes only when elongation is about to begin.A fundamental prerequisite for understanding the biochemical interactions occurring within the ribosome during protein synthesis is a detailed knowledge of the structure of this † Current address: Department of Pharmacology, Weill Medical College, Cornell University, New York, NY 10021 * To whom correspondence should be addressed. Phone (513) 556-1871, Fax (513) Email: Pat.Limbach@uc.edu Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to ou...

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“…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.…”

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confidence: 99%

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

Urlaub

Thiede

Müller

et al. 1997

Journal of Biological Chemistry

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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...

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Protein-rRNA binding features and their structural and functional implications in ribosomes as determined by cross-linking studies.

Cited by 112 publications

References 66 publications

Selecting rRNA binding sites for the ribosomal proteins L4 and L6 from randomly fragmented rRNA: Application of a method called SERF

Selecting rRNA binding sites for the ribosomal proteins L4 and L6 from randomly fragmented rRNA: Application of a method called SERF

Developing limited proteolysis and mass spectrometry for the characterization of ribosome topography

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

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