Structurally conserved Nop56/58 N-terminal domain facilitates archaeal box C/D ribonucleoprotein-guided methyltransferase activity.

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.

Hydrogen bonding and packing density are factors most strongly connected to limiting sites of high flexibility in the 16S rRNA in the 30S ribosome.

BACKGROUND: Conformational flexibility in structured RNA frequently is critical to function. The 30S ribosomal subunit exists in different conformations in different functional states due to changes in the central part of the 16S rRNA. We are interested in evaluating the factors that might be responsible for restricting flexibility to specific parts of the 16S rRNA using biochemical data obtained from the 30S subunit in solution. This problem was approached taking advantage of the observation that there must be a high degree of conformational flexibility at sites where UV photocrosslinking occurs and a lack of flexibility inhibits photoreactivity at many other sites that are otherwise suitable for reaction. RESULTS: We used 30S x-ray structures to quantify the properties of the nucleotide pairs at UV- and UVA-s4U-induced photocrosslinking sites in 16S rRNA and compared these to the properties of many hundreds of additional sites that have suitable geometry but do not undergo photocrosslinking. Five factors that might affect RNA flexibility were investigated - RNA interactions with ribosomal proteins, interactions with Mg2+ ions, the presence of long-range A minor motif interactions, hydrogen bonding and the count of neighboring heavy atoms around the center of each nucleobase to estimate the neighbor packing density. The two factors that are very different in the unreactive inflexible pairs compared to the reactive ones are the average number of hydrogen bonds and the average value for the number of neighboring atoms. In both cases, these factors are greater for the unreactive nucleotide pairs at a statistically very significant level. CONCLUSION: The greater extent of hydrogen bonding and neighbor atom density in the unreactive nucleotide pairs is consistent with reduced flexibility at a majority of the unreactive sites. The reactive photocrosslinking sites are clustered in the 30S subunit and this indicates nonuniform patterns of hydrogen bonding and packing density in the 16S rRNA tertiary structure. Because this analysis addresses inter-nucleotide distances and geometry between nucleotides distant in the primary sequence, the results indicate regional and global flexibility of the rRNA.

Conformational energy and structure in canonical and noncanonical forms of tRNA determined by temperature analysis of the rate of s(4)U8-C13 photocrosslinking.

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.

Internucleotide movements during formation of 16 S rRNA-rRNA photocrosslinks and their connection to the 30 S subunit conformational dynamics.

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.

A 16S rRNA-tRNA product containing a nucleotide phototrimer and specific for tRNA in the P/E hybrid state in the Escherichia coli ribosome.

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.

Pattern of 4-thiouridine-induced cross-linking in 16S ribosomal RNA in the Escherichia coli 30S subunit.

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.

Efficiency and pattern of UV pulse laser-induced RNA-RNA cross-linking in the ribosome.

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.

Conformational change in the 16S rRNA in the Escherichia coli 70S ribosome induced by P/P- and P/E-site tRNAPhe binding.

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.

Action spectra for UV-light induced RNA-RNA crosslinking in 16S ribosomal RNA in the ribosome.

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.