@article{gagnon_biswas_zhang_brown_wollenzien_mattos_maxwell_2012, title={Structurally Conserved Nop56/58 N-terminal Domain Facilitates Archaeal Box C/D Ribonucleoprotein-guided Methyltransferase Activity}, volume={287}, ISSN={["1083-351X"]}, DOI={10.1074/jbc.m111.323253}, abstractNote={Box C/D RNA-protein complexes (RNPs) guide the 2'-O-methylation of nucleotides in both archaeal and eukaryotic ribosomal RNAs. The archaeal box C/D and C'/D' RNP subcomplexes are each assembled with three sRNP core proteins. The archaeal Nop56/58 core protein mediates crucial protein-protein interactions required for both sRNP assembly and the methyltransferase reaction by bridging the L7Ae and fibrillarin core proteins. The interaction of Methanocaldococcus jannaschii (Mj) Nop56/58 with the methyltransferase fibrillarin has been investigated using site-directed mutagenesis of specific amino acids in the N-terminal domain of Nop56/58 that interacts with fibrillarin. Extensive mutagenesis revealed an unusually strong Nop56/58-fibrillarin interaction. Only deletion of the NTD itself prevented dimerization with fibrillarin. The extreme stability of the Nop56/58-fibrillarin heterodimer was confirmed in both chemical and thermal denaturation analyses. However, mutations that did not affect Nop56/58 binding to fibrillarin or sRNP assembly nevertheless disrupted sRNP-guided nucleotide modification, revealing a role for Nop56/58 in methyltransferase activity. This conclusion was supported with the cross-linking of Nop56/58 to the target RNA substrate. The Mj Nop56/58 NTD was further characterized by solving its three-dimensional crystal structure to a resolution of 1.7 Å. Despite low primary sequence conservation among the archaeal Nop56/58 homologs, the overall structure of the archaeal NTD domain is very well conserved. In conclusion, the archaeal Nop56/58 NTD exhibits a conserved domain structure whose exceptionally stable interaction with fibrillarin plays a role in both RNP assembly and methyltransferase activity.}, number={23}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, author={Gagnon, Keith T. and Biswas, Shyamasri and Zhang, Xinxin and Brown, Bernard A., II and Wollenzien, Paul and Mattos, Carla and Maxwell, E. Stuart}, year={2012}, month={Jun}, pages={19418–19428} }
 @article{huggins_ghosh_wollenzien_2009, title={Hydrogen bonding and packing density are factors most strongly connected to limiting sites of high flexibility in the 16S rRNA in the 30S ribosome}, volume={9}, journal={BMC Structural Biology}, author={Huggins, W. and Ghosh, S. K. and Wollenzien, P.}, year={2009} }
 @article{huggins_shapkina_wollenzien_2007, title={Conformational energy and structure in canonical and noncanonical forms of tRNA determined by temperature analysis of the rate of s(4)U8-C13 photocrosslinking}, volume={13}, ISSN={["1469-9001"]}, DOI={10.1261/rna.656907}, abstractNote={Bacterial tRNAs frequently have 4-thiouridine (s(4)U) modification at position 8, which is adjacent to the C13-G22-m(7)G46 base triple in the elbow region of the tRNA tertiary structure. Irradiation with light in the UVA range induces an efficient photocrosslink between s(4)U8 and C13. The temperature dependence of the rate constants for photocrosslinking between the s(4)U8 and C13 has been used to investigate the tRNA conformational energy and structure in Escherichia coli tRNA(Val), tRNA(Phe), and tRNA(fMet) under different conditions. Corrections have been made in the measured rate constants to compensate for differences in the excited state lifetimes due to tRNA identity, buffer conditions, and temperature. The resulting rate constants are related to the rate at which the s(4)U8 and C13 come into the alignment needed for photoreaction; this depends on an activation energy, attributable to the conformational potential energy that occurs during the photoreaction, and on the extent of the structural change. Different photocrosslinking rate constants and temperature dependencies occur in the three tRNAs, and these differences are due both to modest differences in the activation energies and in the apparent s(4)U8-C13 geometries. Analysis of tRNA(Val) in buffers without Mg(2+) indicate a smaller activation energy (~13 kJ mol(-1)) and a larger apparent s(4)U8-C13 distance (~12 A) compared to values for the same parameters in buffers with Mg(2+) (~26 kJ mol(-1) and 0.36 A, respectively). These measurements are a quantitative indication of the strong constraint that Mg(2+) imposes on the tRNA flexibility and structure.}, number={11}, journal={RNA}, author={Huggins, Wayne and Shapkina, Tatjana and Wollenzien, Paul}, year={2007}, month={Nov}, pages={2000–2011} }
 @article{huggins_ghosh_nanda_wollenzien_2005, title={Internucleotide movements during formation of 16 S rRNA-rRNA photocrosslinks and their connection to the 30 S subunit conformational dynamics}, volume={354}, ISSN={["1089-8638"]}, url={http://www.scopus.com/inward/record.url?eid=2-s2.0-27744587220&partnerID=MN8TOARS}, DOI={10.1016/j.jmb.2005.09.060}, abstractNote={UV light-induced RNA photocrosslinks are formed at a limited number of specific sites in the Escherichia coli and in other eubacterial 16 S rRNAs. To determine if unusually favorable internucleotide geometries could explain the restricted crosslinking patterns, parameters describing the internucleotide geometries were calculated from the Thermus thermophilus 30 S subunit X-ray structure and compared to crosslinking frequencies. Significant structural adjustments between the nucleotide pairs usually are needed for crosslinking. Correlations between the crosslinking frequencies and the geometrical parameters indicate that nucleotide pairs closer to the orientation needed for photoreaction have higher crosslinking frequencies. These data are consistent with transient conformational changes during crosslink formation in which the arrangements needed for photochemical reaction are attained during the electronic excitation times. The average structural rearrangement for UVA-4-thiouridine (s4U)-induced crosslinking is larger than that for UVB or UVC-induced crosslinking; this is associated with the longer excitation time for s4U and is also consistent with transient conformational changes. The geometrical parameters do not completely predict the crosslinking frequencies, implicating other aspects of the tertiary structure or conformational flexibility in determining the frequencies and the locations of the crosslinking sites. The majority of the UVB/C and UVA-s4U-induced crosslinks are located in four regions in the 30 S subunit, within or at the ends of RNA helix 34, in the tRNA P-site, in the distal end of helix 28 and in the helix 19/helix 27 region. These regions are implicated in different aspects of tRNA accommodation, translocation and in the termination reaction. These results show that photocrosslinking is an indicator for sites where there is internucleotide conformational flexibility and these sites are largely restricted to parts of the 30 S subunit associated with ribosome function.}, number={2}, journal={JOURNAL OF MOLECULAR BIOLOGY}, author={Huggins, W and Ghosh, SK and Nanda, K and Wollenzien, P}, year={2005}, month={Nov}, pages={358–374} }
 @article{huggins_wollenzien_2004, title={A 16S rRNA-tRNA product containing a nucleotide phototrimer and specific for tRNA in the P/E hybrid state in the Escherichia coli ribosome}, volume={32}, ISSN={["1362-4962"]}, DOI={10.1093/nar/gkh1001}, abstractNote={Ribosome complexes containing deacyl-tRNA1(Val) or biotinylvalyl-tRNA1(Val) and an mRNA analog have been irradiated with wavelengths specific for activation of the cmo5U nucleoside at position 34 in the tRNA1(Val) anticodon loop. The major product for both types of tRNA is the cross-link between 16S rRNA (C1400) and the tRNA (cmo5U34) characterized already by Ofengand and his collaborators [Prince et al. (1982) Proc. Natl Acad. Sci. USA, 79, 5450-5454]. However, in complexes containing deacyl-tRNA1(Val), an additional product is separated by denaturing PAGE and this is shown to involve C1400 and m5C967 of 16S rRNA and cmo5U34 of the tRNA. Puromycin treatment of the biotinylvalyl-tRNA1(Val) -70S complex followed by irradiation, results in the appearance of the unusual photoproduct, which indicates an immediate change in the tRNA interaction with the ribosome after peptide transfer. These results indicate an altered interaction between the tRNA anticodon and the 30S subunit for the tRNA in the P/E hybrid state compared with its interaction in the classic P/P state.}, number={22}, journal={NUCLEIC ACIDS RESEARCH}, author={Huggins, W and Wollenzien, P}, year={2004}, pages={6548–6556} }
 @article{shapkina_lappi_franzen_wollenzien_2004, title={Efficiency and pattern of UV pulse laser-induced RNA-RNA cross-linking in the ribosome}, volume={32}, ISSN={["1362-4962"]}, DOI={10.1093/nar/gkh320}, abstractNote={Escherichia coli ribosomes were irradiated with a KrF excimer laser (248 nm, 22 ns pulse) with incident pulse energies in the range of 10-40 mJ for a 1 cm2 area, corresponding to fluences of 4.5 to 18 x 10(9) W m(-2), to determine strand breakage yields and the frequency and pattern of RNA-RNA cross- linking in the 16S rRNA. Samples were irradiated in a cuvette with one laser pulse or in a flow cell with an average of 4.6 pulses per sample. The yield of strand breaks per photon was intensity dependent, with values of 0.7 to 1.3 x 10(-3) over the incident intensity range studied. The yield for RNA-RNA cross-linking was 3 x 10(-4) cross-links/photon at the intensity of 4.5 x 10(9) W m(-2), an approximately 4-fold higher yield per photon than obtained with a transilluminator. The cross-link yield/photon decreased at higher light intensities, probably due to intensity-dependent photoreversal. The pattern of cross-linking was similar to that observed with low intensity irradiation but with four additional long-range cross-links not previously seen in E.coli ribosomes. Cross- linking frequencies obtained with one laser pulse are more correlated to internucleotide distances than are frequencies obtained with transilluminator irradiation.}, number={4}, journal={NUCLEIC ACIDS RESEARCH}, author={Shapkina, T and Lappi, S and Franzen, S and Wollenzien, P}, year={2004}, month={Feb}, pages={1518–1526} }
 @article{nanda_wollenzien_2004, title={Pattern of 4-thiouridine-induced cross-linking in 16S ribosomal RNA in the Escherichia coli 30S subunit}, volume={43}, ISSN={["0006-2960"]}, DOI={10.1021/bi049702h}, abstractNote={The locations of RNA-RNA cross-links in 16S rRNA were determined after in vivo incorporation of 4-thiouridine (s(4)U) into RNA in a strain of Escherichia coli deficient in pyrimidine synthesis and irradiation at >320 nm. This was done as an effort to find RNA cross-links different from UVB-induced cross-links that would be valuable for monitoring the 30S subunit in functional complexes. Cross-linked 16S rRNA was separated on the basis of loop size, and cross-linking sites were identified by reverse transcription, RNase H cleavage, and RNA sequencing. A limited number of RNA-RNA cross-links in nine regions were observed. In five regions-s(4)U562 x C879-U884, s(4)U793 x A1519, s(4)U1189 x U1060-G1064, s(4)U1183 x A1092, and s(4)U991 x C1210-U1212-the s(4)U-induced cross-links are similar to UVB-induced cross-links observed previously. In four other regions-s(4)U960 x A1225, s(4)U820 x G570, s(4)U367 x A55-U56, and s(4)U239 x A120-the s(4)U-induced cross-links are different from UVB-induced cross-links. The pattern of cross-linking is not limited by the distribution of s(4)U, because there are at least 112 s(4)U substitution sites in the 16S rRNA. The relatively small number of s(4)U-mediated cross-links is probably determined by the organization of the RNA in the 30S subunit, which allows RNA conformational flexibility needed for cross-link formation in just a limited region.}, number={28}, journal={BIOCHEMISTRY}, author={Nanda, K and Wollenzien, P}, year={2004}, month={Jul}, pages={8923–8934} }
 @article{zhirnov_wollenzien_2003, title={Action spectra for UV-light induced RNA-RNA crosslinking in 16S ribosomal RNA in the ribosome}, volume={2}, ISSN={["1474-9092"]}, DOI={10.1039/b208677h}, abstractNote={UV irradiation induces intramolecular crosslinks in ribosomal RNA in the ribosome. These crosslinks occur between nucleotides distant in primary sequence and they are specific, limited in number and have crosslinking efficiencies sufficient to allow their use in monitoring conformational changes. In this work, the frequency of crosslinking for eight 16S rRNA crosslinks was determined as a function of wavelength of irradiation. For six of the crosslinks, the action spectra correspond to the absorption spectra of at least one of the participating nucleotides. For a crosslink between nucleotides C967 and C1400 the maximum frequency of crosslinking occurs at wavelengths blue-shifted from the absorbance maximum of cytidine and for a crosslink between C1402 and C1501 the maximum frequency of crosslinking is red-shifted. Photoreversal of the crosslinks was also studied by deproteinizing crosslinked RNA under mild conditions and then re-irradiating it with specific wavelengths under conditions in which the crosslinks were reversed but not formed. The different crosslinks exhibit significantly different extents of photoreversal versus wavelength profiles. The differences in the crosslinking action spectra can be accounted for in the absorbance spectra of the nucleotides that are involved in the crosslink as well as by the photoreversal action spectra.}, number={6}, journal={PHOTOCHEMICAL & PHOTOBIOLOGICAL SCIENCES}, author={Zhirnov, OV and Wollenzien, P}, year={2003}, pages={688–693} }
 @article{noah_shapkina_nanda_huggins_wollenzien_2003, title={Conformational change in the 16S rRNA in the Escherichia coli 70S ribosome induced by P/P- and P/E-site tRNA(Phe) binding}, volume={42}, ISSN={["0006-2960"]}, DOI={10.1021/bi035369q}, abstractNote={The effects of P/P- and P/E-site tRNA(Phe) binding on the 16S rRNA structure in the Escherichia coli 70S ribosome were investigated using UV cross-linking. The identity and frequency of 16S rRNA intramolecular cross-links were determined in the presence of deacyl-tRNA(Phe) or N-acetyl-Phe-tRNA(Phe) using poly(U) or an mRNA analogue containing a single Phe codon. For N-acetyl-Phe-tRNA(Phe) with either poly(U) or the mRNA analogue, the frequency of an intramolecular cross-link C967 x C1400 in the 16S rRNA was decreased in proportion to the binding stoichiometry of the tRNA. A proportional effect was true also for deacyl-tRNA(Phe) with poly(U), but the decrease in the C967 x C1400 frequency was less than the tRNA binding stoichiometry with the mRNA analogue. The inhibition of the C967 x C1400 cross-link was similar in buffers with, or without, polyamines. The exclusive participation of C967 with C1400 in the cross-link was confirmed by RNA sequencing. One intermolecular cross-link, 16S rRNA (C1400) to tRNA(Phe)(U33), was made with either poly(U) or the mRNA analogue. These results indicate a limited structural change in the small subunit around C967 and C1400 during tRNA P-site binding sensitive to the type of mRNA that is used. The absence of the C967 x C1400 cross-link in 70S ribosome complexes with tRNA is consistent with the 30S and 70S crystal structures, which contain tRNA or tRNA analogues; the occurrence of the cross-link indicates an alternative arrangement in this region in empty ribosomes.}, number={49}, journal={BIOCHEMISTRY}, author={Noah, JW and Shapkina, TG and Nanda, K and Huggins, W and Wollenzien, P}, year={2003}, month={Dec}, pages={14386–14396} }
 @article{juzumiene_wollenzien_2001, title={Arrangement of the central pseudoknot region of 16S rRNA in the 30S ribosomal subunit determined by site-directed 4-thiouridine crosslinking}, volume={7}, ISSN={["1469-9001"]}, DOI={10.1017/S1355838201001728}, abstractNote={The 16S rRNA central pseudoknot region in the 30S ribosomal subunit has been investigated by photocrosslinking from 4-thiouridine (s4U) located in the first 20 nt of the 16S rRNA. RNA fragments (nt 1-20) were made by in vitro transcription to incorporate s4U at every uridine position or were made by chemical synthesis to incorporate s4U into one of the uridine positions at +5, +14, +17, or +20. These were ligated to RNA containing nt 21-1542 of the 16S rRNA sequence and, after gel purification, the ligated RNA was reconstituted into 30S subunits. Long-range intramolecular crosslinks were produced by near-UV irradiation; these were separated by gel electrophoresis and analyzed by reverse transcription reactions. A number of crosslinks are made in each of the constructs, which must reflect the structural flexibility or conformational heterogeneity in this part of the 30S subunit. All of the constructs show crosslinking to the 559-562, 570-571, and 1080-1082 regions; however, other sites are crosslinked specifically from each s4U position. The most distinctive crosslinking sites are: 341-343 and 911-917 for s4U(+5); 903-904 (very strong), 1390-1397, and 1492 for s4U(+14); and 903-904 (moderate) for s4U(+17); in the 1070-1170 region in which there are different patterns for each s4U position. These results indicate that part of the central pseudoknot is in close contact with the decoding region, with helix 27 in the 885-912 interval and with part of domain III RNA. Crosslinking between s4U(+14) and 1395-1397 is consistent with base pairing at U14-A1398.}, number={1}, journal={RNA}, author={Juzumiene, DI and Wollenzien, P}, year={2001}, month={Jan}, pages={71–84} }
 @article{dolan_babin_wollenzien_2001, title={Construction and analysis of base-paired regions of the 16S rRNA in the 30S ribosomal subunit determined by constraint satisfaction molecular modelling}, volume={19}, ISSN={["1873-4243"]}, DOI={10.1016/S1093-3263(00)00097-8}, abstractNote={Structure models for each of the secondary structure regions from the Escherichia coli 16S rRNA (58 separate elements) were constructed using a constraint satisfaction modelling program to determine which helices deviated from classic A-form geometry. Constraints for each rRNA element included the comparative secondary structure, H-bonding conformations predicted from patterns of base-pair covariation, tertiary interactions predicted from covariation analysis, chemical probing data, rRNA-rRNA crosslinking information, and coordinates from solved structures. Models for each element were built using the MC-SYM modelling algorithm and subsequently were subjected to energy minimization to correct unfavorable geometry. Approximately two-thirds of the structures that result from the input data are very similar to A-form geometry. In the remaining instances, the presence of internal loops and bulges, some sequences (and sequence covariants) and accessory information require deviation from A-form geometry. The structures of regions containing more complex base-pairing arrangements including the central pseudoknot, the 530 region, and the pseudoknot involving base-pairing between G570-U571/A865-C866 and G861-C862/G867-C868 were predicted by this approach. These molecular models provide insight into the connection between patterns of H-bonding, the presence of unpaired nucleotides, and the overall geometry of each element.}, number={6}, journal={JOURNAL OF MOLECULAR GRAPHICS & MODELLING}, author={Dolan, MA and Babin, P and Wollenzien, P}, year={2001}, pages={495–513} }
 @article{juzumiene_shapkina_kirillov_wollenzien_2001, title={Short-range RNA-RNA crosslinking methods to determine rRNA structure and interactions}, volume={25}, ISSN={["1046-2023"]}, DOI={10.1006/meth.2001.1245}, abstractNote={We describe details of procedures to analyze RNA-RNA crosslinks made by far-UV irradiation (< 300 nm) or made by irradiation with near-UV light (320-365 nm) on RNA containing photosensitive nucleotides, in the present case containing 4-thiouridine. Zero-length crosslinks of these types must occur because of the close proximity of the participants through either specific interactions or transient contacts in the folded RNA structure, so they are valuable monitors of the conformation of the RNA. Procedures to produce crosslinks in the 16S ribosomal RNA and between the 16S rRNA and mRNA or tRNA are described. Gel electrophoresis conditions are described that separate the products according to their structure to allow the determination of the number and frequency of the crosslinking products. Gel electrophoresis together with an ultracentrifugation procedure for the efficient recovery of RNA from the polyacrylamide gels allows the purification of molecules containing different crosslinks. These separation techniques allow the analysis of the sites of crosslinking by primer extension and RNA sequencing techniques. The procedures are applicable to other types of RNA molecules with some differences to control levels of crosslinking and separation conditions.}, number={3}, journal={METHODS}, author={Juzumiene, D and Shapkina, T and Kirillov, S and Wollenzien, P}, year={2001}, month={Nov}, pages={333–343} }
 @article{shapkina_dolan_babin_wollenzien_2000, title={Initiation factor 3-induced structural changes in the 30 S ribosomal subunit and in complexes containing tRNA(f)(Met) and mRNA}, volume={299}, ISSN={["1089-8638"]}, DOI={10.1006/jmbi.2000.3774}, abstractNote={Initiation factor 3 (IF3) acts to switch the decoding preference of the small ribosomal subunit from elongator to initiator tRNA. The effects of IF3 on the 30 S ribosomal subunit and on the 30 S.mRNA. tRNA(f)(Met) complex were determined by UV-induced RNA crosslinking. Three intramolecular crosslinks in the 16 S rRNA (of the 14 that were monitored by gel electrophoresis) are affected by IF3. These are the crosslinks between C1402 and C1501 within the decoding region, between C967xC1400 joining the end loop of a helix of 16 S rRNA domain III and the decoding region, and between U793 and G1517 joining the 790 end loop of 16 S rRNA domain II and the end loop of the terminal helix. These changes occur even in the 30 S.IF3 complex, indicating they are not mediated through tRNA(f)(Met) or mRNA. UV-induced crosslinks occur between 16 S rRNA position C1400 and tRNA(f)(Met) position U34, in tRNA(f)(Met) the nucleotide adjacent to the 5' anticodon nucleotide, and between 16 S rRNA position C1397 and the mRNA at positions +9 and +10 (where A of the initiator AUG codon is +1). The presence of IF3 reduces both of these crosslinks by twofold and fourfold, respectively. The binding site for IF3 involves the 790 region, some other parts of the 16 S rRNA domain II and the terminal stem/loop region. These are located in the front bottom part of the platform structure in the 30 S subunit, a short distance from the decoding region. The changes that occur in the decoding region, even in the absence of mRNA and tRNA, may be induced by IF3 from a short distance or could be caused by the second IF3 structural domain.}, number={3}, journal={JOURNAL OF MOLECULAR BIOLOGY}, author={Shapkina, TG and Dolan, MA and Babin, P and Wollenzien, P}, year={2000}, month={Jun}, pages={615–628} }
 @article{juzumiene_wollenzien_2000, title={Organization of the 16S rRNA around its 5 ' terminus determined by photochemical crosslinking in the 30S ribosomal subunit}, volume={6}, ISSN={["1469-9001"]}, DOI={10.1017/S1355838200991659}, abstractNote={The organization of the 5' terminus region in the 16S rRNA was investigated using a series of RNA constructs in which the 5' terminus was extended by 5 nt or was shortened to give RNA molecules that started at positions -5, +1, +5, +8, +14, or +21. The structural and functional effects of the 5' extension/truncations were determined after the RNAs were reconstituted. 30S subunits containing 16S rRNA with 5' termini at -5, +1, +5, +8 and +14 had similar structures (judged by UV-induced crosslinking) and exhibited a gradual reduction in tRNA binding activity compared to that seen with 30S subunits reconstituted with native 16S rRNA. To create the 5' terminal site-specific photocrosslinking agent, the reagent azidophenacylbromide (APAB) was attached to the 5' terminus of 16S rRNA through a guanosine monophosphorothioate and the APA-16S rRNAs were reconstituted. Crosslinking carried out with the APA revealed sites in six regions around positions 300-340, 560, 900, 1080, the 16S rRNA decoding region, and at 1330. Differences in the pattern and efficiency of crosslinking for the different constructs allow distance estimates for the crosslinked sites from nucleotide G9. These measurements provide constraints for the arrangement of the RNA elements in the 30S subunit. Similar experiments carried out in the 70S ribosome resulted in a five- to tenfold lower frequency of crosslinking. This is most likely due to a repositioning of the 5' terminus upon subunit association.}, number={1}, journal={RNA}, author={Juzumiene, DI and Wollenzien, P}, year={2000}, month={Jan}, pages={26–40} }
 @inbook{mundus_wollenzien_2000, title={Structure determination by directed photo-cross-linking in large RNA molecules with site-specific psoralen}, volume={318}, DOI={10.1016/s0076-6879(00)18047-4}, booktitle={RNA-ligland interactions: Part B}, publisher={San Diego, CA: Academic Press}, author={Mundus, D. and Wollenzien, P.}, editor={R.W. Skaggs and Schilfgaarde, J.Editors}, year={2000}, pages={104–118} }
 @article{noah_shapkina_wollenzien_2000, title={UV-induced crosslinks in the 16S rRNAs of Escherichia coli, Bacillus subtilis and Thermus aquaticus and their implications for ribosome structure and photochemistry}, volume={28}, ISSN={["1362-4962"]}, DOI={10.1093/nar/28.19.3785}, abstractNote={Sixteen long-range crosslinks are induced in Escherichia coli 16S rRNA by far-UV irradiation. Crosslinking patterns in two other organisms, Bacillus subtilis and Thermus aquaticus, were investigated to determine if the number and location of crosslinks in E.coli occur because of unusually photoreactive nucleotides at particular locations in the rRNA sequence. Thirteen long-range crosslinks in B.subtilis and 15 long-range crosslinks in T.aquaticus were detected by gel electrophoresis and 10 crosslinks in each organism were identified completely by reverse transcription analysis. Of the 10 identified crosslinks in B.subtilis, eight correspond exactly to E.coli crosslinks and two crosslinks are formed close to sites of crosslinks in E.coli. Of the 10 identified crosslinks in T.aquaticus, five correspond exactly to E.coli crosslinks, three are formed close to E.coli crosslinking sites, one crosslink corresponds to a UV laser irradiation-induced crosslink in E.coli and the last is not seen in E.coli. The overall similarity of crosslink positions in the three organisms suggests that the crosslinks arise from tertiary interactions that are highly conserved but with differences in detail in some regions.}, number={19}, journal={NUCLEIC ACIDS RESEARCH}, author={Noah, JW and Shapkina, T and Wollenzien, P}, year={2000}, month={Oct}, pages={3785–3792} }
 @article{noah_dolan_babin_wollenzien_1999, title={Effects of tetracycline and spectinomycin on the tertiary structure of ribosomal RNA in the Escherichia coli 30 S ribosomal subunit}, volume={274}, ISSN={["0021-9258"]}, DOI={10.1074/jbc.274.23.16576}, abstractNote={Structural analysis of the 16 S rRNA in the 30 S subunit and 70 S ribosome in the presence of ribosome-specific antibiotics was performed to determine whether they produced rRNA structural changes that might provide further insight to their action. An UV cross-linking procedure that determines the pattern and frequency of intramolecular 16 S RNA cross-links was used to detect differences reflecting structural changes. Tetracycline and spectinomycin have specific effects detected by this assay. The presence of tetracycline inhibits the cross-link C967×C1400 completely, increases the frequency of cross-link C1402×1501 twofold, and decreases the cross-link G894×U244 by one-half without affecting other cross-links. Spectinomycin reduces the frequency of the cross-link C934×U1345 by 60% without affecting cross-linking at other sites. The structural changes occur at concentrations at which the antibiotics exert their inhibitory effects. For spectinomycin, the apparent binding site and the affected cross-linking site are distant in the secondary structure but are close in tertiary structure in several recent models, indicating a localized effect. For tetracycline, the apparent binding sites are significantly separated in both the secondary and the three-dimensional structures, suggesting a more regional effect. Structural analysis of the 16 S rRNA in the 30 S subunit and 70 S ribosome in the presence of ribosome-specific antibiotics was performed to determine whether they produced rRNA structural changes that might provide further insight to their action. An UV cross-linking procedure that determines the pattern and frequency of intramolecular 16 S RNA cross-links was used to detect differences reflecting structural changes. Tetracycline and spectinomycin have specific effects detected by this assay. The presence of tetracycline inhibits the cross-link C967×C1400 completely, increases the frequency of cross-link C1402×1501 twofold, and decreases the cross-link G894×U244 by one-half without affecting other cross-links. Spectinomycin reduces the frequency of the cross-link C934×U1345 by 60% without affecting cross-linking at other sites. The structural changes occur at concentrations at which the antibiotics exert their inhibitory effects. For spectinomycin, the apparent binding site and the affected cross-linking site are distant in the secondary structure but are close in tertiary structure in several recent models, indicating a localized effect. For tetracycline, the apparent binding sites are significantly separated in both the secondary and the three-dimensional structures, suggesting a more regional effect. A variety of antibiotics interact with the ribosome to inhibit protein synthesis within bacterial and eukaryotic cells (1Gale E.F. Cundliffe E. Reynolds P.E. Richmond M.H. Waring M.J. The Molecular Basis of Antibiotic Action. Wiley, London1981Google Scholar). The point of interruption in the translation cycle has been determined for some antibiotics (2Spahn C.M.T. Prescott C.D. J. Mol. Med. 1996; 74: 423-439Crossref PubMed Scopus (173) Google Scholar), making them useful in vitro to investigate the nature of elongation in protein synthesis. Some of these antibiotics have been footprinted on the ribosomal RNAs in 30 S and 50 S subunits and in the 70 S ribosome (3Moazed D. Noller H.F. Nature. 1987; 327: 389-394Crossref PubMed Scopus (967) Google Scholar) as well as on model oligomers designed to mimic local regions of 16 S rRNA (4Purohit P. Stern S. Nature. 1994; 370: 659-662Crossref PubMed Scopus (255) Google Scholar, 5Recht M.I. Fourmy D. Blanchard S.C. Dahlquist K.D. Puglisi J.D. J. Mol. Biol. 1996; 262: 421-436Crossref PubMed Scopus (235) Google Scholar). Sites of action have also been established by affinity and photoaffinity experiments (6Cooperman B.S. Press U.P. Ribosomes Structure, Function, and Genetics. University Park Press, New York1980: 531-554Google Scholar, 7Oehler R. Polacek N. Steiner G. Barta A. Nucleic Acids Res. 1997; 25: 1219-1224Crossref PubMed Scopus (51) Google Scholar). The binding sites frequently correspond to regions of the ribosome that are implicated by other experiments in ribosome function. In many cases, binding site assignments are supported by resistance-conferring mutations in 16 S and 23 S rRNAs (2Spahn C.M.T. Prescott C.D. J. Mol. Med. 1996; 74: 423-439Crossref PubMed Scopus (173) Google Scholar, 8Triman K.L. Adv. Genet. 1995; 33: 1-37Crossref PubMed Scopus (11) Google Scholar). Antibiotics, either directly by binding or indirectly through conformation alterations, must result in the inability of tRNA or translation factors to bind the ribosome or else inhibit some processes needed during translation. However, little is known regarding whether conformational perturbations accompany binding, even in instances in which detailed information supporting binding sites and the nature of translation interruption are known. UV cross-linking of the rRNA in the ribosome provides an opportunity to monitor changes in rRNA conformation and, consequently, the ribosome global structure. We have previously determined the identity of 14 UV-induced cross-links in 16 S rRNA within the 30 S subunit (9Wilms C. Noah J.W. Zhong D. Wollenzien P. RNA. 1997; 3: 602-612PubMed Google Scholar) and 15 cross-links in the 70 S ribosome (10Noah J. Wollenzien P. Biochemistry. 1998; 37: 15442-15448Crossref PubMed Scopus (15) Google Scholar). Because of the gel electrophoresis method used in the detection, all of these cross-links occur between nucleotides that are distant in the primary sequence. These cross-links occur because the partner nucleotides possess a suitable distance and geometry during the lifetime of the excited state (on the order of 1 μs; Ref. 11Budowsky E.I. Axentyeva M.S. Abdurachidova G.G. Simukova N.A. Rubin L.B. Eur. J. Biochem. 1989; 159: 95-101Crossref Scopus (34) Google Scholar); therefore, their frequency provides a method to screen substrates and other agents for their ability to affect ribosomal conformation. In this report, UV irradiation was repeated in the presence of 13 antibiotics to determine whether they produce measurable changes in the ribosome structure that might be related to their activity. These 13 antibiotics were known to bind either the 30 S or 50 S subunit. Some of these antibiotics have localized binding sites on 16 S rRNA, as determined by chemical probing, that are adjacent in the 16 S rRNA secondary structure to nucleotides that participate in cross-links and may affect the frequency of such contacts. Two of these antibiotics, spectinomycin and tetracycline, have discernible effects on the frequency of specific UV cross-linking sites in 16 S rRNA. The implications of these effects with respect to the antibiotic action are discussed. Escherichia coli 70 S ribosomes and ribosomal subunits were prepared according to Makhno et al. (12Makhno V.I. Peshin N.N. Semenkov Y.P. Kirillov S.V. Mol. Biol. 1988; 22: 528-537Google Scholar) and dissolved in CMN 1The abbreviations used are: CMN, 80 mm cacodylic acid, pH 7.5, 20 mmMgCl2, 100 mm NH4Cl, and 4 mm β-mercaptoethanol; BTBE, 30 mmbis(2-hydroxyethyl)iminotris(hydroxymethyl)methane, 30 mmboric acid, and 2.5 mm EDTA, pH 6.8. buffer. In some experiments, CMN buffer was used with Mg2+concentrations from 0.5 to 50 mm. 70 S ribosomes were prepared by reassociating equimolar amounts of 30 S and 50 S subunits and were free of mRNA or tRNA. Samples were incubated with the following concentrations of antibiotics (Sigma) that were previously determined to produce specific footprints in the 16 S rRNA or 23 S rRNA by chemical probing (2Spahn C.M.T. Prescott C.D. J. Mol. Med. 1996; 74: 423-439Crossref PubMed Scopus (173) Google Scholar, 3Moazed D. Noller H.F. Nature. 1987; 327: 389-394Crossref PubMed Scopus (967) Google Scholar): (a) 5 × 10−6m neomycin, (b) 5 × 10−6m paromomycin, (c) 5 × 10−6m streptomycin, (d) 1 × 10−4m gentamycin, (e) 1 × 10−4m kanamycin, (f) 1 × 10−4m spectinomycin, (g) 2.5 × 10−4m tetracycline, (h) 5 × 10−6m erythromycin, (i) 5 × 10−6m thiostrepton, (j) 5 × 10−6m fusidic acid, (k) 5 × 10−6m chloramphenicol, (l) 5 × 10−6m viomycin, and (m) 1 × 10−4m hygromycin. Concentration series have also been investigated for tetracycline and spectinomycin. Samples were incubated for 30 min at 37 °C, placed on ice for 10 min, and irradiated at 4 °C for 20 min in a quartz cuvette with continuous stirring. Irradiation was performed with a 312 nm trans-illuminator (Fotodyne Corp.) as described previously (9Wilms C. Noah J.W. Zhong D. Wollenzien P. RNA. 1997; 3: 602-612PubMed Google Scholar). Sample concentrations were usually 1 μg RNA/μl, with the exception of samples for preparative separation, which were irradiated at 6 μg RNA/μl. RNA was recovered from the samples by proteinase K digestion, phenol extraction, and ethanol precipitation. The RNA was dephosphorylated with shrimp intestinal phosphatase and purified by proteinase K digestion, phenol extraction, and ethanol precipitation. 16 S rRNA was then isolated on a 1% agarose gel before 5′ end-labeling with [γ-32P]ATP by T4 polynucleotide kinase. Cross-linked 16 S rRNA was separated by gel electrophoresis on gels made with 3.6% acrylamide:bisacrylamide (70:1), 8.3 murea, and BTBE buffer as described previously (9Wilms C. Noah J.W. Zhong D. Wollenzien P. RNA. 1997; 3: 602-612PubMed Google Scholar). For analysis of cross-linking sites, the location of the bands containing un-cross-linked and cross-linked 16 S rRNA were detected with a PhosphorImager, and bands were cut out and eluted by ultracentrifugation through cushions containing 2 m CsCl and 0.2 m EDTA, pH 7.4, for 12 h at 40,000 rpm (13Wilms C. Wollenzien P. Anal. Biochem. 1994; 221: 204-205Crossref PubMed Scopus (16) Google Scholar). RNA pellets were redissolved in 250 μl of H20, phenol-extracted, and re-precipitated before further analysis. The cross-linking sites in separated 16 S rRNA were found by primer extension analysis using 11 DNA primers complementary to regions throughout 16 S rRNA (9Wilms C. Noah J.W. Zhong D. Wollenzien P. RNA. 1997; 3: 602-612PubMed Google Scholar). The frequency of cross-linking was determined from PhosphorImager data (ImageQuant; Molecular Dynamics Inc.) of duplicate independent experiments. To normalize for RNA loading, cross-link band intensity was referenced to the same cross-link band (C54×A353) in each respective lane. This reference band showed <10% variance in all lanes when referenced to the un-cross-linked 16 S rRNA parent band in the same lane. Six classes of antibiotics were examined in these experiments: (a) aminoglycoside (neomycin, paromomycin, streptomycin, gentamycin, kanamycin, spectinomycin, and hygromycin), (b) tetracycline, (c) macrolide (erythromycin), (d) peptide (thiostrepton and viomycin), (e) fusidic acid, and (f) chloramphenicol. The seven aminoglycosides, tetracycline, and the two peptide antibiotics are thought to bind primarily to the 30 S subunit, and the two peptide antibiotics also bind to the 50 S subunit (2Spahn C.M.T. Prescott C.D. J. Mol. Med. 1996; 74: 423-439Crossref PubMed Scopus (173) Google Scholar). Erythromycin and chloramphenicol bind exclusively to the 50 S subunit, and fusidic acid prevents the release of elongation factor G·GDP from the ribosome (2Spahn C.M.T. Prescott C.D. J. Mol. Med. 1996; 74: 423-439Crossref PubMed Scopus (173) Google Scholar). These last three antibiotics were included to test the possibility that an alteration in the 50 S subunit structure may induce changes in the structure of the 30 S subunit, within the 70 S ribosome. Both 30 S and 70 S ribosomes were irradiated in the presence of the listed antibiotics, and, with exceptions noted below, the effects were identical. In all instances in these experiments, empty ribosomes were used to avoid heterogeneity in the ribosomal state and to avoid the complication of possible substrate-mediated structure changes. In addition, previous characterizations of antibiotic binding sites (3Moazed D. Noller H.F. Nature. 1987; 327: 389-394Crossref PubMed Scopus (967) Google Scholar, 7Oehler R. Polacek N. Steiner G. Barta A. Nucleic Acids Res. 1997; 25: 1219-1224Crossref PubMed Scopus (51) Google Scholar) and our identification of UV cross-links in 16 S rRNA (9Wilms C. Noah J.W. Zhong D. Wollenzien P. RNA. 1997; 3: 602-612PubMed Google Scholar) have been performed on empty ribosomes. Tetracycline and spectinomycin were the only compounds tested that affected the cross-linking pattern of 16 S rRNA within 30 S subunits or 70 S ribosomes (Fig. 1). At 2.5 × 10−4m, the concentration used for chemical footprinting experiments (3Moazed D. Noller H.F. Nature. 1987; 327: 389-394Crossref PubMed Scopus (967) Google Scholar), tetracycline affected three identified cross-links and one incompletely identified cross-link. The third band from the top of the pattern, which was verified as C967×C1400, was completely inhibited. In this and other gels, there was some decrease in the intensity of the topmost band in the pattern, which was shown to contain U244×G894. The third band from the bottom of the gel, which was shown to contain the cross-link C1402×C1501, increased twofold in intensity in both 30 S subunits and 70 S ribosomes. The fourth band from the top also decreased in the presence of tetracycline. U534 is one part of this cross-link, but we have been unable to find a partner for it. At 1 × 10−4m, the concentration used for chemical footprinting experiments (3Moazed D. Noller H.F. Nature. 1987; 327: 389-394Crossref PubMed Scopus (967) Google Scholar), spectinomycin decreased the intensity of the cross-link found in the second band from the top (Fig. 1), which was shown to contain the cross-link C934×U1345, by 60%. Reverse transcription analyses of the regions of 16 S rRNA containing the cross-links affected by tetracycline are shown in Fig.2. A and B show the results of reverse transcription in the nucleotide intervals 212–265 and 866–914, respectively, in which differences were seen in the tetracycline sample. In lane 7 in each panel, the stops indicating cross-links at U244 and G894 decreased in the tetracycline sample relative to the control sample. C and Dshow the results of reverse transcription reactions in the nucleotide intervals 952–983 and 1387–1409. Lane 5 of each panel contains stops indicating that the cross-link C967×C1400 disappeared.E and F show the results of reverse transcription reactions in the nucleotide intervals 1390–1411 and 1491–1504.Lane 2 of each panel contains stops indicating that the cross-link C1402×C1501 increased in the presence of tetracycline relative to the control. The effects of tetracycline on cross-link intensity are the same in both 30 S subunits and 70 S ribosomes. Reverse transcription analysis of the cross-linked band affected by spectinomycin is shown in Fig. 3. The only reverse transcription stops affected by spectinomycin were in intervals 907–953 (A) and 1330–1365 (B). A decrease in the stops at 935 and 1346 in lane 6 of each panel indicates that the cross-link C934×1345 is affected. The decreases in intensity are consistent with the difference seen in the analytical gel pattern (Fig. 1). Titration experiments were performed with tetracycline to determine the concentration threshold on the 16 S rRNA tertiary structure (Fig.4). At concentrations between 2.5 × 10−4m and 1 × 10−5m, tetracycline specifically affects the cross-links noted above. There was no change in the frequency of any of the cross-links before the low concentration limit was reached. In the sample irradiated in the highest concentration of tetracycline, several cross-links were partially or completely inhibited; this is attributed to the nonspecific binding of tetracycline to the ribosome and/or a decrease in cross-linking due to the high tetracycline absorbance. Titration experiments were also performed at five spectinomycin concentrations (Fig. 4). These show that the decrease in the C934×U1345 cross-link is seen at or above 1 × 10−6m spectinomycin, with a complete recovery of cross-link frequency below 1 × 10−6m. The effects of tetracycline and spectinomycin were determined in 30 S subunits and 70 S ribosomes at Mg2+ concentrations from 0.5 to 50 mm. Mg2+ has been shown to govern the frequency of cross-linking of several cross-links including C967×C1400 and C1402×C1501 (10Noah J. Wollenzien P. Biochemistry. 1998; 37: 15442-15448Crossref PubMed Scopus (15) Google Scholar). C967×C1400 was inhibited at all Mg2+ concentrations, except that in 0.5 mmMg2+ in 30 S ribosomes, the frequency of the cross-link, even in the control sample, was too low to detect. The increase in the frequency of C1402×C1501 due to tetracycline was seen in 30 S subunits and 70 S ribosomes at all Mg2+ concentrations above 5 mm. Below 5 mm, no increase in C1402×C1501 could be seen (data not shown). The decrease in frequency of C934×U1345 due to spectinomycin did not change over the stated Mg2+ concentration range (data not shown). Several cross-links, C1400×C1501, C1402×C1501, and C1397×C1497, identified in the decoding region of 16 S rRNA nearly co-migrate in gel electrophoresis (10Noah J. Wollenzien P. Biochemistry. 1998; 37: 15442-15448Crossref PubMed Scopus (15) Google Scholar). There was a possibility that neomycin and streptomycin would affect the distribution of conformations in that region. However, no detectable changes in the analytical gel electrophoresis experiments involving these antibiotics were seen, nor were any changes seen in primer extension experiments (data not shown). Of the 13 antibiotics examined in this study, tetracycline and spectinomycin showed specific and different structural effects in 16 S rRNA that were detectable by the UV cross-linking assay. The inhibition of cross-links by these antibiotics correlates well with their known effective concentrations (14Semenkov Y.P. Makarov E.M. Makhno V.I. Kirillov S.V. FEBS Lett. 1982; 144: 125-129Crossref PubMed Scopus (36) Google Scholar, 15Dragon F. Spickler C. Pinard R. Carriere J. Brakier-Gingras L. J. Mol. Biol. 1996; 259: 207-215Crossref PubMed Scopus (9) Google Scholar), indicating that the 16 S rRNA tertiary structure effects are linked to the loss of small subunit function during translation. For the tetracycline response, all three of the cross-links are affected at the same tetracycline concentration. Tetracycline has been shown to inhibit protein synthesis by interfering with the binding of aminoacyl-tRNA to the ribosomal A-site (2Spahn C.M.T. Prescott C.D. J. Mol. Med. 1996; 74: 423-439Crossref PubMed Scopus (173) Google Scholar, 14Semenkov Y.P. Makarov E.M. Makhno V.I. Kirillov S.V. FEBS Lett. 1982; 144: 125-129Crossref PubMed Scopus (36) Google Scholar), but it does not prevent the binding of tRNAPhe to the P-site (16Wurmbach P. Nierhaus K.H. Eur. J. Biochem. 1983; 130: 9-12Crossref PubMed Scopus (26) Google Scholar). However, tetracycline has been reported to interfere with initiation factor-dependent tRNAfMet binding (2Spahn C.M.T. Prescott C.D. J. Mol. Med. 1996; 74: 423-439Crossref PubMed Scopus (173) Google Scholar, 17Sarkar S. Thaach R.E. Proc. Natl. Acad. Sci. U. S. A. 1968; 60: 1479-1486Crossref PubMed Scopus (53) Google Scholar). In a study in which the binding of a fluorescent analogue of tetracycline, demeclocycline, was monitored, displacement of demeclocycline by tRNAfMet and by A-site tRNAPhebinding was confirmed (18Epe B. Woolley P. Hornig H. FEBS Lett. 1987; 213: 443-447Crossref PubMed Scopus (39) Google Scholar). Tetracycline did have an inhibitory effect on the P-site when the determination was done at high tRNA:70 S stoichiometries, and this was attributed to a fraction of ribosomes that bind tRNA in the P-site with lower affinity (19Geigenmuller U. Nierhaus K.H. Eur. J. Biochem. 1986; 161: 723-726Crossref PubMed Scopus (37) Google Scholar). Photochemical cross-linking experiments have shown that tetracycline cross-links primarily to protein S 7 and, to a lesser extent, S 18 and S 4 (20Goldman R.A. Hasan T. Hall C.C. Strycharz W.A. Cooperman B.S. Biochemistry. 1983; 22: 359-368Crossref PubMed Scopus (104) Google Scholar). S 7 has been footprinted by both base modification and Fe2+/EDTA cleavage experiments to the lower part of domain III in 16 S rRNA (21Powers T. Noller H.F. RNA. 1995; 1: 194-209PubMed Google Scholar). More recent experiments have determined that with tetracycline concentrations lower than 4 × 10−5m, higher UV light flux, and shorter irradiation times (30 s), tetracycline cross-linked to 16 S rRNA itself at nucleotides G693 (near helix 23b), G1300, and G1338 (both of them near helix 42) (7Oehler R. Polacek N. Steiner G. Barta A. Nucleic Acids Res. 1997; 25: 1219-1224Crossref PubMed Scopus (51) Google Scholar). At concentrations of ≥1.2 × 10−4m, additional cross-linking sites, including one at G890, are seen (7Oehler R. Polacek N. Steiner G. Barta A. Nucleic Acids Res. 1997; 25: 1219-1224Crossref PubMed Scopus (51) Google Scholar). In experiments done at 1 × 10−4m, tetracycline produced a strong chemical protection footprint in 16 S rRNA at nucleotide A892 and a weak reactivity enhancement of nucleotides U1052 and C1054 (3Moazed D. Noller H.F. Nature. 1987; 327: 389-394Crossref PubMed Scopus (967) Google Scholar) (see Fig.5). Recently, a C→G substitution at position 1058 granting resistance to tetracycline was reported (22Ross J.I. Eady E.A. Cunliffe W.J. Antimicrob. Agents Chemother. 1998; 42: 1702-1705Crossref PubMed Google Scholar), which also indicates that this region is connected to tetracycline function. Footprints for tRNA in the P-site include C1400, G1338, C967, and G693 (23Moazed D. Noller H.F. J. Mol. Biol. 1990; 211: 135-145Crossref PubMed Scopus (249) Google Scholar). Because two of the three affected UV cross-links and all three tetracycline-RNA cross-links are associated with the tRNA P binding site, it is worthwhile to consider how tetracycline might interfere with A-site tRNA binding. Rodnina et. al. (24Rodnina M.V. Fricke R. Wintermeyer W. Biochemistry. 1994; 33: 12267-12275Crossref PubMed Scopus (125) Google Scholar) have investigated EFTu·aminoacyl tRNA·GTP binding and found that the rate and strength of the initial complex and the codon recognition complex were both enhanced by cognate tRNA bound to the P-site. This suggests an effect by the P-site-bound tRNA or some change in the ribosome that depends on P-site occupancy that influences the ternary complex-ribosome interaction. Therefore, alteration of the P-site by tetracycline may not affect the binding of tRNA in the P-site itself but may inhibit some conformational adjustment needed for tRNA binding to the A-site. The reported antibiotic binding sites (3Moazed D. Noller H.F. Nature. 1987; 327: 389-394Crossref PubMed Scopus (967) Google Scholar, 7Oehler R. Polacek N. Steiner G. Barta A. Nucleic Acids Res. 1997; 25: 1219-1224Crossref PubMed Scopus (51) Google Scholar) and the affected RNA cross-linking sites are compared in a 16 S rRNA three-dimensional model (Fig.6), 2M. A. Dolan, P. Babin, and P. Wollenzien, unpublished data. which shows the locations of the helices containing the tetracycline and spectinomycin binding sites and the RNA-RNA cross-links affected by their binding. This model is different from recent models (26Mueller F. Brimacombe R. J. Mol. Biol. 1997; 271: 524-544Crossref PubMed Scopus (120) Google Scholar, 27Malhotra A. Harvey S.C. J. Mol. Biol. 1994; 240: 308-340Crossref PubMed Scopus (109) Google Scholar, 28Fink D.L. Chen R.O. Noller H.F. Altman R.B. RNA. 1996; 2: 851-866PubMed Google Scholar) in several regions because it incorporates new information including the UV cross-links C967×C1400 (9Wilms C. Noah J.W. Zhong D. Wollenzien P. RNA. 1997; 3: 602-612PubMed Google Scholar, 10Noah J. Wollenzien P. Biochemistry. 1998; 37: 15442-15448Crossref PubMed Scopus (15) Google Scholar), U793×G1517, and G976×G1361 3T. Shapkina, unpublished data. and site-specific psoralen cross-links (25Mundus D. Wollenzien P. RNA. 1998; 4: 1373-1385Crossref PubMed Scopus (17) Google Scholar). 4D. Mundus, unpublished data. Approximate distances from G1300, G1338, and G693 to C1400 (the P-site) are 45, 35, and 25 Å, respectively, and distances from G1300, G1338, and G693 to A1408 (associated with the A-site) are 57, 50, and 36 Å, respectively. In another recently proposed model (26Mueller F. Brimacombe R. J. Mol. Biol. 1997; 271: 524-544Crossref PubMed Scopus (120) Google Scholar), the distances to C1400 are 67, 40, and 81Å, and the distances to A1408 are 95, 70, and 82 Å. In both models, the distances between the sites of tetracycline-RNA cross-linking (7Oehler R. Polacek N. Steiner G. Barta A. Nucleic Acids Res. 1997; 25: 1219-1224Crossref PubMed Scopus (51) Google Scholar) and the P-site nucleotide C1400 (3Moazed D. Noller H.F. Nature. 1987; 327: 389-394Crossref PubMed Scopus (967) Google Scholar) are less than those to the A-site nucleotide A1408 (23Moazed D. Noller H.F. J. Mol. Biol. 1990; 211: 135-145Crossref PubMed Scopus (249) Google Scholar). The molecular dimensions of tetracycline measure approximately 8 × 12 Å. Spectinomycin causes a structural change in domain III that results in a decrease in the frequency of cross-link C934×U1345. Spectinomycin causes a strong protection from chemical reactivity at C1063 and G1064 (see Fig. 5) and a weak enhancement of reactivity at G973 (3Moazed D. Noller H.F. Nature. 1987; 327: 389-394Crossref PubMed Scopus (967) Google Scholar). In addition, spectinomycin resistance is conferred by a C→A, G, U mutation at position 1192 in 16 S rRNA. Because overexpression of a fragment of helix 34 corresponding to nucleotides 1047–1067 and 1189–1210 (linked by a hairpin loop) also confers spectinomycin resistance (2Spahn C.M.T. Prescott C.D. J. Mol. Med. 1996; 74: 423-439Crossref PubMed Scopus (173) Google Scholar), the binding site is most likely located in helix 34. In addition, it was shown that susceptibility to spectinomycin could be restored in C1192 mutants by an additional U1351→C mutation in the top of helix 43 (15Dragon F. Spickler C. Pinard R. Carriere J. Brakier-Gingras L. J. Mol. Biol. 1996; 259: 207-215Crossref PubMed Scopus (9) Google Scholar). The partial inhibition of cross-link C934×U1345 by spectinomycin supports evidence for an interplay between helix 34b and 43. The helix that contains the spectinomycin footprint at C1063 (helix 34b) and the nucleotides that form the C934×U1345 cross-link at the end of helix 28 are highlighted in the model (Fig. 6). The distance between these two regions is 12 Å in the model shown (Fig. 6), compared with 20 Å in the Mueller and Brimacombe (26Mueller F. Brimacombe R. J. Mol. Biol. 1997; 271: 524-544Crossref PubMed Scopus (120) Google Scholar) model. A surprising result is that spectinomycin has no effect on the U1052×C1200 cross-link, which is in close proximity to both the resistance mutation site (C1192) (3Moazed D. Noller H.F. Nature. 1987; 327: 389-394Crossref PubMed Scopus (967) Google Scholar) and the proposed binding site in 16 S rRNA. In addition, there are no changes in the cross-links U1052×C1200, A1093×G1182, or U1126×C1281, all of which are located in domain III. This indicates that spectinomycin is not producing a global rearrangement of the domain III tertiary structure but rather some specific alteration of the structure near the junctions of helices 28, 29, and 43. It is known that spectinomycin interferes with elongation factor G binding during early rounds of protein synthesis (2Spahn C.M.T. Prescott C.D. J. Mol. Med. 1996; 74: 423-439Crossref PubMed Scopus (173) Google Scholar). It has recently been shown that elongation factor G interacts with the 30 S subunit near the 1338 region (29Wilson K.S. Noller H.F. Cell. 1998; 92: 131-139Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar); therefore, it also possible that the structural perturbation caused by spectinomycin alters elongation factor G association. The neomycin-type aminoglycosides (hygromycin, gentamycin, neomycin, and paromomycin) protect 16 S rRNA nucleotides A790, A791, A909, A1394, A1413, and G1487 and enhance reactivity at C525 (3Moazed D. Noller H.F. Nature. 1987; 327: 389-394Crossref PubMed Scopus (967) Google Scholar). These antibiotics are generally thought to induce miscoding by inhibiting A-site occupation and to inhibit translocation (2Spahn C.M.T. Prescott C.D. J. Mol. Med. 1996; 74: 423-439Crossref PubMed Scopus (173) Google Scholar). Streptomycin causes miscoding (2Spahn C.M.T. Prescott C.D. J. Mol. Med. 1996; 74: 423-439Crossref PubMed Scopus (173) Google Scholar) and protects 16 S rRNA nucleotides 911–915, nucleotides in the 1408–1418 and 1482–1494 regions, and nucleotide 1468 in 30 S subunits in 20 mm Mg2+ (3Moazed D. Noller H.F. Nature. 1987; 327: 389-394Crossref PubMed Scopus (967) Google Scholar, 30Spickler C. Brunelle M. Brakier-Gingras L. J. Mol. Biol. 1997; 273: 586-599Crossref PubMed Scopus (29) Google Scholar) and binds to naked 16 S rRNA fragments of the decoding region in 20 mmMg2+. Viomycin and thiostrepton induce protections in both 16 S and 23 S rRNA (for viomycin, these are known to be protections at nucleotides 912–915), and both antibiotics also inhibit A-site occupation and cause miscoding (2Spahn C.M.T. Prescott C.D. J. Mol. Med. 1996; 74: 423-439Crossref PubMed Scopus (173) Google Scholar). It is thought that their binding restricts the ribosome conformation, thereby preventing A-site occupation or translocation (2Spahn C.M.T. Prescott C.D. J. Mol. Med. 1996; 74: 423-439Crossref PubMed Scopus (173) Google Scholar). None of those antibiotics produces an observable effect in this experiment, so it is possible that these antibiotics interact with only a local region of the ribosome. Alternatively, the tertiary structure effects for the other antibiotics may be too subtle to detect by the present cross-linking, may be elicited only in the ribosome under working conditions, or may affect parts of the ribosome not monitored by the cross-links that are made by UV irradiation. We thank Vickers Burdett for comments on the manuscript.}, number={23}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, author={Noah, JW and Dolan, MA and Babin, P and Wollenzien, P}, year={1999}, month={Jun}, pages={16576–16581} }
 @article{babin_dolan_wollenzien_gutell_1999, title={Identity and geometry of a base triple in 16S rRNA determined by comparative sequence analysis and molecular modeling}, volume={5}, ISSN={["1469-9001"]}, DOI={10.1017/S1355838299990659}, abstractNote={Comparative sequence analysis complements experimental methods for the determination of RNA three-dimensional structure. This approach is based on the concept that different sequences within the same gene family form similar higher-order structures. The large number of rRNA sequences with sufficient variation, along with improved covariation algorithms, are providing us with the opportunity to identify new base triples in 16S rRNA. The three-dimensional conformations for one of our strongest candidates involving U121 (C124:G237) and/or U121 (U125:A236) (Escherichia coli sequence and numbering) are analyzed here with different molecular modeling tools. Molecular modeling shows that U121 interacts with C124 in the U121 (C124:G237) base triple. This arrangement maintains isomorphic structures for the three most frequent sequence motifs (approximately 93% of known bacterial and archaeal sequences), is consistent with chemical reactivity of U121 in E. coli ribosomes, and is geometrically favorable. Further, the restricted set of observed canonical (GU, AU, GC) base-pair types at positions 124:237 and 125:236 is consistent with the fact that the canonical base-pair sets (for both base pairs) that are not observed in nature prevent the formation of the 121 (124:237) base triple. The analysis described here serves as a general scheme for the prediction of specific secondary and tertiary structure base pairing where there is a network of correlated base changes.}, number={11}, journal={RNA}, author={Babin, P and Dolan, M and Wollenzien, P and Gutell, RR}, year={1999}, month={Nov}, pages={1430–1439} }
 @article{noah_wollenzien_1998, title={Dependence of the 16S rRNA decoding region structure on Mg2+, subunit association, and temperature}, volume={37}, ISSN={["0006-2960"]}, DOI={10.1021/bi981148m}, abstractNote={The effects of Mg2+ concentration, subunit association, and temperature on the structure of 16S rRNA in the Escherichia coli ribosome were investigated using UV cross-linking and gel electrophoresis analysis. Mg2+ concentrations between 1 and 20 mM and temperatures between 5 and 55 degreesC had little effect on the frequency of 12 of the 14 cross-links in 30S subunits and modest effects on the same cross-links in 70S ribosomes. In contrast, two cross-links, C967 x C1400 and C1402 x C1501, involving rRNA in the decoding region are present in 30S subunits only above 3 mM Mg2+, increase in frequency at higher Mg2+ concentration, and are both more frequent when 50S subunits are included in the reactions. In 70S ribosomes, the cross-link C1402 x C1501 increases but the cross-link C967 x C1400 decreases at higher Mg2+ concentrations. One cross-link, C1397 x U1495, is detected only in 70S ribosomes and decreases in frequency as Mg2+ concentration is increased. An additional cross-link, A1093 x C1182, decreases upon subunit association. The cross-link frequency differences indicate that the arrangement of the decoding region of the 16S rRNA, but not in the rest of the subunit, is readily altered by Mg2+ ions and subunit association.}, number={44}, journal={BIOCHEMISTRY}, author={Noah, JW and Wollenzien, P}, year={1998}, month={Nov}, pages={15442–15448} }
 @article{mundus_wollenzien_1998, title={Neighborhood of 16S rRNA nucleotides U788/U789 in the 30S ribosomal subunit determined by site-directed crosslinking}, volume={4}, ISSN={["1469-9001"]}, DOI={10.1017/S1355838298981134}, abstractNote={Site-specific photo crosslinking has been used to investigate the RNA neighborhood of 16S rRNA positions U788/ U789 in Escherichia coli 30S subunits. For these studies, site-specific psoralen (SSP) which contains a sulfhydryl group on a 17 A side chain was first added to nucleotides U788/U789 using a complementary guide DNA by annealing and phototransfer. Modified RNA was purified from the DNA and unmodified RNA. For some experiments, the SSP, which normally crosslinks at an 8 A distance, was derivitized with azidophenacylbromide (APAB) resulting in the photoreactive azido moiety at a maximum of 25 A from the 4' position on psoralen (SSP25APA). 16S rRNA containing SSP, SSP25APA or control 16S rRNA were reconstituted and 30S particles were isolated. The reconstituted subunits containing SSP or SSP25APA had normal protein composition, were active in tRNA binding and had the usual pattern of chemical reactivity except for increased kethoxal reactivity at G791 and modest changes in four other regions. Irradiation of the derivatized 30S subunits in activation buffer produced several intramolecular RNA crosslinks that were visualized and separated by gel electrophoresis and characterized by primer extension. Four major crosslink sites made by the SSP reagent were identified at positions U561/U562, U920/U921, C866 and U723; a fifth major crosslink at G693 was identified when the SSP25APA reagent was used. A number of additional crosslinks of lower frequency were seen, particularly with the APA reagent. These data indicate a central location close to the decoding region and central pseudoknot for nucleotides U788/U789 in the activated 30S subunit.}, number={11}, journal={RNA}, author={Mundus, D and Wollenzien, P}, year={1998}, month={Nov}, pages={1373–1385} }
 @article{wilms_noah_zhong_wollenzien_1997, title={Exact determination of UV-induced crosslinks in 16S ribosomal RNA in 30s ribosomal subunits}, volume={3}, number={6}, journal={RNA}, author={Wilms, C. and Noah, J. W. and Zhong, D. G. and Wollenzien, P. L.}, year={1997}, pages={602–612} }
 @article{wilms_noah_zhong_wollenzien_1997, title={Exact determination of UV-induced crosslinks in 16S ribosomal RNA in 30s ribosomal subunits: Erratum}, volume={3}, number={8}, journal={RNA}, author={Wilms, C. and Noah, J. W. and Zhong, D. and Wollenzien, P.}, year={1997}, pages={945} }