Structure and accessibility of domain I of Escherichia coli 23 S RNA in free RNA, in the L24-RNA complex and in 50 S subunits

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

“…A: Secondary structure of the E. coli 295-343 region+ Bases in capitals are conserved between E. coli and H. marismortui, small letters indicate E. coli sequence+ The U between G301 and C302 indicates the additional nucleotide in H. marismortui. Iodine cleavage results with the ternary L24-rRNA L4 -L4 complex are shown as in Figure 2, but omitting the arrows that indicated increased binding+ The color code in B and C is the same concerning the RNA backbone and the protection sites+ B: Three-dimensional structure of the RNA backbone from the X-ray structure of the H. marismortui 50S subunit (PDB: 1FFK; Ban et al+, 2000)+ C: The proteins L4 and L24 bind the rRNA L4 region from two different sites (a-C backbone of L24 and L4 in grey; Ban et al+, 2000)+ The L4 interaction site G319-C323 (H. m.: G326-C330) is shown in red, and that for L24 in green (G298-G301) and cyan (C337-G338), that is, C304-G307 (H. m.) and C344-G345 (H. m.), respectively (Ban et al+, 2000)+ In-gel probing results of the L24-rRNA L4 -L4 ternary complex are indicated+ Pro-R P oxygen positions, that were protected are shown as spheres according to the color code: G297-A300 in green, A309-A310 and C314-G315 in orange, A330 and C336 in blue, G337-G338 in cyan, U339 and A340 in violet, and A320-C323 in red, except the interference site A322, which is indicated as a yellow sphere+ pair (A507-C483 of H. m.) located in H24 of domain I distant in the secondary structure map+ The presence of protein L24 at this RNA-RNA interaction side agrees well with L24-dependent protections of bases in the 500 region of domain I (site B in Egebjerg et al+, 1987)+ We further note that A309 (A316 H. m.) in the loop of H19 base pairs with G1210 (U1314 H. m.) in H46 of domain II and that protein L4 has additional contacts to other rRNA strands, in particular to domain II (Ban et al+, 2000)+…”

“…The role of L24 in organizing the folding the 23S rRNA is also indicated by the fact that this proteins renders the whole domain I (comprising the sequence 1-560) largely nuclease resistant (Zimmermann, 1980) and stabilizes the structure of this domain (Egebjerg et al+, 1987)+ Although L24 has additional RNA contacts in domain I and L4 is elongated, contacting domains II and V (Gregory & Dahlberg, 1999;Ban et al+, 2000), we suggest that the rRNA fragment examined here is one essential trigger for the far-reaching allosteric actions of proteins L24 and L4 on the rRNA folding during early assembly events+…”

“…A comparison of the cleavage pattern of noncomplexed RNA extracted from the gel and then cleaved under denaturing conditions (7 M urea) with that of in-gel cleavage of noncomplexed RNA under native conditions reveals only minor differences (compare the second and fourth lanes in Fig+ 4)+ Evidently, the RNA under gel-shift conditions is either not strongly structured or the structure only moderately affects the iodine accessibility+ There is one surprising exception, namely position U293, that is not part of the minimal binding site for the proteins (Stelzl et al+, 2000a) and cannot be cleaved within the gel+ In the E. coli 23S rRNA secondary structure, U293 is directly 59 to an asymmetric internal loop in H18 that consists solely of adenines, and evidently its structure prevents access of iodine to the 59 phosphate group of U293+ This residue is not conserved+ Helix 18 is very variable among different species (Egebjerg et al+, 1987); for example, an adenine rich internal loop in H18 does not exist in the archaea Haloarcula marismortui (Ban et al+, 2000)+ The regions of the rRNA fragment presumably interacting with the proteins directly match perfectly in a comparison of the probing results of the two techniques, namely, isolation of the complexes from nitrocellulose filter (Fig+ 2B;Stelzl et al+, 2000a) and in-gel cleavage (Fig+ 2C, red and green nucleotides for L4 and L24, respectively)+ This fact gives credit to the ingel cleavage variant and allows a precise definition of the interaction sites+ The L4 binding site is presumably represented by the U321 loop, that is, the nucleotides G319-A322 (Stelzl et al+, 2000a), and this conclusion is supported by the a Position indicates the phosphate 59 to the nucleotide under focus+ Bold positions: the relative intensity of the band differs by a factor of 1+5 from the intensity of the corresponding band of the compared cleavage pattern (i+e+, relative intensities Յ0+66 and Ն1+50; these positions are indicated in Fig+ 2D)+ b Interference indicates intensities of the positions of the RNA extracted from the complex in the gel and cleaved after extraction, divided by the corresponding intensities of an RNA that was subjected to electrophoresis as free RNA and cleaved after extraction from the gel+ c Protection indicates intensities of the RNA bands from in-gel cleaved complexes divided by the intensities of the corresponding bands of the RNA extracted from the complex in the gel and cleaved after extraction+ The range of experimental values are indicated as largest deviation (6) from the average of five independent experiments made with two different RNA preparations+ We note that four errors in the interference column (G313, G315, A320, and U321) are higher than all others+ This is partially due to the high absolute values of the signals+ following observations+ (1) Phosphorothioates 59 and 39 to U321 impede binding+ (2) The two upstream residues (G319 and A320) are protected from cleavage in the L4-rRNA complex+ (3) A 2-nt deletion of C316 and G317 right next to the site was reported that abolishes L4 action in an in vitro competition assay (Zengel & Lindahl, 1993)+…”

Incorporating chemical modification constraints into a dynamic programming algorithm for prediction of RNA secondary structure

“…This limited specificity differs from that observed with nucleases, and an algorithm allowing constraints from such chemical modification has not been reported. Chemical modification is used extensively to test hypothesized RNA secondary structures (19,(23)(24)(25)(26)(27)(28). Chemical modification can also be used to deduce possible tertiary contacts within an RNA (29), to probe RNA bound to protein (25, 26, 30 -35), or to follow RNA folding pathways (36 -38).…”

Puromycin–rRNA interaction sites at the peptidyl transferase center

“…gibbonsii occur at the universally conserved nt G2505 and G2553+ G2505 is the site of altered nucleotide reactivity in the presence of several other antibiotics, including the macrolides carbomycin and tylosin, and the streptogramin A drugs )+ In a recent investigation, G2553 was specifically crosslinked to the A-site tRNA analog, 4-thio-dT-p-C-p-puromycin (Green et al+, 1998)+ In another study, all possible mutations made at G2553 in E. coli resulted in dominant growth defects in vivo, as well as reduced peptidyl transferase activity in vitro (Kim & Green, 1999)+ In addition, complementation analysis of mutant A-site substrate analogs and ribosomes established a specific interaction between C75 of the aminoacyl-tRNA and G2553 of 23S rRNA (Kim & Green, 1999)+ An important role for this rRNA loop in puromycin binding is also suggested by mutagenesis and damage-selection experiments, in which changes at 2550, 2552, 2555, and 2557 interfered with the transfer FIGURE 6. Putative allosteric effects dependent on puromycin in domains I and III of E. coli 23S rRNA+ Nucleotides exhibiting enhanced reactivities in the presence of puromycin are boxed in (A) domain I and (B) domain III+ The shaded regions are binding sites for ribosomal proteins L24 (Egebjerg et al+, 1987) and L23 (Egebjerg et al+, 1991)+ FIGURE 7. The effect of puromycin on the crosslinking yields of deacylated (2N 3 A76)tRNA Phe , bound at the ribosomal P site, to fragments of 23S rRNA+ Complexes were prepared in the absence (control) or presence of puromycin (1 mM), before binding of [ 32 P](2N 3 A76)tRNA Phe and irradiation (see Materials and methods)+ The specific activity of [ 32 P](2N 3 A76)tRNA Phe was 3,000 dpm/pmol+ The crosslinked fragments were generated by RNase H digestion with a set of deoxyoligonucleotides (see Materials and methods), and analyzed on polyacrylamide gels+ The positions of the three main labeled fragments F19, F29, and F49 are indicated+ Although the intensity of the F19 crosslink appears reduced in the puromycin lane, when the counts are adjusted for the degree of tRNA binding to ribosomes, the differences are minimal ( none 13 6 2 2 5 6 3 7 6 1 puromycin 10 6 1 1 3 6 2 n + b + a Data are presented as counts per minute of crosslinked deacylated (2N 3 A76)tRNA Phe per picomole of ribosome-bound deacylated (2N 3 A76)tRNA Phe + The specific activity of the (2N 3 A76)tRNA Phe was normalized to an initial activity of 10,000 dpm/pmol or about 1,800 cpm/pmol estimated in an Instant Imager+ The data were averaged from four separate experiments+ a n+b+: no band detected+ of peptidyl moiety to puromycin Bocchetta et al+, 1998)+ Taken together, the data suggest that the loop containing G2553 folds into the catalytic center and is in close proximity with the 39 end of the aminoacyl-tRNA substrate+ The region between 2058 and 2062 of the peptidyltransferase loop is an area of diverse reactivity changes for many antibiotics and is known to be conformationally labile+ In the case of puromycin, there is strong protection of G2058 (G2084) in the halophiles but no effect at A2058 in E. coli+ Differences in the chemical footprint of the streptogramin A drugs, bound to archaeal and bacterial ribosomes, have also been observed at this position (Rodriguez-Fonseca et al+, 1995; )+ In the presence of pristinamycin IIA, protection at G2058 (G2084) is observed in Halobacteria halobium, whereas enhancements are seen at A2058 in Bacillus megaterium, both in vitro and in vivo )+ The conformational flexibility of this region is underscored by the variable weak effects at position 2062 that were observed in E. col...…”