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

Abstract

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...

A short fragment of 23S rRNA containing the binding sites for two ribosomal proteins, L24 and L4, is a key element for rRNA folding during early assembly

“…The effects of phosphorothioate modification on RNAprotein interaction match direct structural information obtained from corresponding X-ray structures or that derived by cryoelectron microscopy very well+ For example, results obtained with the MS2 coat protein-RNA (Milligan & Uhlenbeck, 1989;Dertinger et al+, 2000) and tRNA-synthetase systems (Schatz et al+, 1991;Rudinger et al+, 1992;Voertler et al+, 1998) are directly comparable to interactions seen in three-dimensional crystal structures of the complexes+ Phosphorothioated tRNAs bound by the ribosome give specific cleavage patterns that are characteristic for the particular ribosomal binding site (Dabrowski et al+, 1995) and that are dependent on the functional state of the ribosome (Dabrowski et al+, 1998)+ The protection pattern observed with P-site bound peptidyl-tRNA analogs was in perfect agreement with the ribosome contacts seen with an f-Met-tRNA at the ribosomal P site in cryoelectron microscopy pictures (Malhotra et al+, 1998)+ Recently, we selected a short fragment of domain I of 23S rRNA (GG295-343CC, 53 nt) that binds independently and simultaneously the ribosomal proteins L4 and L24 (Stelzl et al+, 2000a(Stelzl et al+, , 2000b, two key proteins for the early assembly of the large subunit of bacterial ribosomes (Nierhaus, 1991)+ L24 is one of the two assembly initiator proteins of the 50S subunit (Nowotny & Nierhaus, 1982), and L4 is essential for the formation of the first assembly intermediate particle (Spillmann et al+, 1977)+ Here, we present phosphorothioate probing data of the ternary L24-rRNA-L4 complex determined by an in-gel analysis and compare them with that of the two binary complexes L24-rRNA and rRNA-L4+ In-gel iodine cleavage enables the probing of RNA structures in homogeneous populations of the different complexes, thereby overcoming the problem that a preparation of a ternary complex still contains a fraction of binary complexes obscuring the signals+ The results demonstrate that the rRNA region within the binary complexes adopts a conformation that is different from that observed in the ternary complex+ We discuss these probing data in light of the recent crystallographically determined three-dimensional model of the 50S ribosomal subunit (Ban et al+, 2000)+…”

“…Both ribosomal proteins L4 and L24 bind independently and simultaneously to a 53-nt rRNA fragment of domain I in 23S rRNA (GGG295-C343CC) with mM Ϫ1 affinities (Stelzl et al+, 2000a)+ The rRNA in the two corresponding binary complexes was characterized by phosphorothioate cleavage; the complexes were separated from the nonbound rRNA fragments by nitrocellulose filtration+…”

“…In the following, we describe the binding of the two proteins, L4 and L24, to the fragment rRNA L4 with a length of 77 nt (Fig+ 2A)+ The initial G of this GG283-U358 fragment (rRNA L4-2 in Stelzl et al+, 2000a) is not derived from the rRNA sequence, but is due to transcription requirements+ We do not use the minimal 53-nt fragment (rRNA L4-3 in Stelzl et al+, 2000a), because the corresponding complexes with the longer RNA L4 fragment separate well in gel electrophoresis (Fig+ 1B), significantly better than the complexes with the smaller rRNA fragment (not shown)+ Both fragments have the same protein binding capabilities (Stelzl et al+, 2000a)+ Nitrocellulose filtration is a nonequilibrium method for measuring binding affinities, the separation of the complex from the noncomplexed molecules requires several seconds+ The respective situation in gel-shift experiments is more harsh, because the electrophoresis lasts 2 h under our conditions, and weakly bound RNA molecules are removed from the complex+ As a consequence, the apparent association constants are usually smaller (up to one order of magnitude), when measured via gel shift as compared to nitrocellulose filter measurement (Draper et al+, 1988)+ It follows that the more stringent method, namely, the gel-shift experiment, changes the pattern in the direction of higher binding selectivity+ This is indicated by the following observations+ (1) When we compare the filtration results of the two binary complexes with those of in-gel cleavage, clearly more O-to-S substitutions affect bind- to G307, G308 in H19, A320 and 59 to A332 and G333 in H20+ In the in-gel cleavage experiment, a thioate at G301 interferes with L4 binding (red stars)+ (2) In the band-shift experiment, four positions with a phosphorothioate modification were enriched in the complexes FIGURE 2. A: Secondary structure of the 59 half of E. coli 23S rRNA (1-1646)+ The rRNA L4 fragment used is indicated in red (GG283-U358, 77 nt)+ B-D: Iodine cleavage pattern projected onto the rRNA secondary structure+ Intensities of bands that differ by a factor of 1+5 from the comparison are indicated: ‫ء‬ decreased and F increased binding (from interference experiments); ᭹ decreased and ࡗ enhanced cleavage in the complex (from protection experiments)+ B: Cleavage patterns of the binary complexes as seen with the nitrocellulose filter technique, L24-rRNA in green and rRNA-L4 in red; A G298U (green nucleotide) mutation abolishes L24 binding (Stelzl et al+, 2000a)+ C: Cleavage patterns as seen with in-gel cleavage technique; nucleotide positions interacting with L24 and L4 are highlighted in green and red, respectively+ D: In-gel probing results of the L24-rRNA L4--L4 ternary complex (blue)+ (arrows in Fig+ 2C), that is, the substitutions favor the binding of the protein as compared to the nonmodified molecules+ An oxygen-to-sulfur substitution at A300 and A322 stimulates binding of L24 (green arrows); a similar effect is seen for positions A320 and C331 in the case of protein L4 (red arrows)+ Protection experiments reflect the accessibility of the RNA backbone for iodine within the RNA-protein complex+ Some protections seen in the filter assays do not show up in the gel-shift assay (e+g+, circles around A330 in Fig+ 2B but not in 2C), and a few protection signals are even replaced by interference signals (A309, G312, and A320 in the case of L24, A299, and C318 for L4) in the gel-shift approach+ Binding of L24 exposes A311 and G338 to I 2 cleavage in the complex within the gel (Fig+ 2C, diamonds)+ Based on the nitrocellulose filter probing results in the previous analysis …”

“…A: Secondary structure of the 59 half of E. coli 23S rRNA (1-1646)+ The rRNA L4 fragment used is indicated in red (GG283-U358, 77 nt)+ B-D: Iodine cleavage pattern projected onto the rRNA secondary structure+ Intensities of bands that differ by a factor of 1+5 from the comparison are indicated: ‫ء‬ decreased and F increased binding (from interference experiments); ᭹ decreased and ࡗ enhanced cleavage in the complex (from protection experiments)+ B: Cleavage patterns of the binary complexes as seen with the nitrocellulose filter technique, L24-rRNA in green and rRNA-L4 in red; A G298U (green nucleotide) mutation abolishes L24 binding (Stelzl et al+, 2000a)+ C: Cleavage patterns as seen with in-gel cleavage technique; nucleotide positions interacting with L24 and L4 are highlighted in green and red, respectively+ D: In-gel probing results of the L24-rRNA L4--L4 ternary complex (blue)+ (arrows in Fig+ 2C), that is, the substitutions favor the binding of the protein as compared to the nonmodified molecules+ An oxygen-to-sulfur substitution at A300 and A322 stimulates binding of L24 (green arrows); a similar effect is seen for positions A320 and C331 in the case of protein L4 (red arrows)+ Protection experiments reflect the accessibility of the RNA backbone for iodine within the RNA-protein complex+ Some protections seen in the filter assays do not show up in the gel-shift assay (e+g+, circles around A330 in Fig+ 2B but not in 2C), and a few protection signals are even replaced by interference signals (A309, G312, and A320 in the case of L24, A299, and C318 for L4) in the gel-shift approach+ Binding of L24 exposes A311 and G338 to I 2 cleavage in the complex within the gel (Fig+ 2C, diamonds)+ Based on the nitrocellulose filter probing results in the previous analysis (Stelzl et al+, 2000a), the RNA regions directly interacting with the proteins were tentatively assigned+ Despite the apparent differences of the patterns caused by the two techniques, the probing data within these regions are exactly the same+ The L4 interaction site is the sequence G319-A322 (red in Fig+ 2C)+ The phosphate backbone connections 59 to G319 and A320 are protected from cleavage in the complex and the O-to-S backbone substitutions at U321 and A322 impede binding of L4+ The cleavage pattern of this region is shown in Figure 3A+ The L24 interaction site is located in both the G298-G301 and the C337-G338 region (green in Fig+ 2C)+ The cleavage pattern in the first region is the same in both approaches, that is, G298 and A300 are strongly protected from iodine cleavage by L24, and phosphorothioates at A299, G301, and C302 interfere with binding of the protein (this gel section is shown in Fig+ 3B)+ In the second region, however, the pattern is changed: the G338 substitution is underrepresented but exposed to cleavage in the complex, and the C334-C337 signal is reduced in the complex with L24+…”

Accommodating the bacterial decoding release factor as an alien protein among the RNAs at the active site of the ribosome

“…How might the subtleties of these footprints be elucidated biochemically? As a start to resolving this problem, we have trialled a modified SELEX procedure that had been used successfully to define more precisely known binding sites of specific ribosomal proteins with rRNA [48][49][50] after fragmentation of the rRNA into short sequences. Importantly, the binding sites of these proteins with intact ribosomes were reproduced from the fragmented rRNA, showing that the technique could identify physiologically relevant short binding motifs.…”