The topography of the 3''-terminal region of Escherichia coli 16S ribosomal RNA; an intra-RNA cross-linking study.

30S ribosomal subunits, 70S ribosomes or polysomes from E. coli were subjected to mild ultraviolet irradiation, and the 3'-terminal region of the 16S RNA was excised by 'addressed cleavage' using ribonuclease H in the presence of suitable complementary oligodeoxynucleotides. RNA fragments from this region containing intra-RNA cross-links were separated by two-dimensional gel electrophoresis and the cross-link sites identified by our standard procedures. Five new cross-links were found in the 30S subunit, which were localized at positions 1393-1401 linked to 1531-1532, 1393-1401 linked to 1506, 1393-1401 to 1502-1504, 1402-1403 to 1498-1501, and 1432 to 1465-69, respectively. In 70S ribosomes or polysomes the first four of these were absent, but instead two cross-links between the 1400-region and tRNA were observed. These results are discussed in the context of the tertiary folding of the 3'-terminal region of the 16S RNA and its known functional significance as part of the ribosomal decoding centre.     

Probing dynamic changes in rRNA conformation in the 30S subunit of the Escherichia coli ribosome.

Ribosomal RNA molecules within each ribosomal subunit are folded in a specific three-dimensional form. The accessibility of specific sequences of rRNA of the small ribosomal subunit of Escherichia coli was analyzed using complementary oligodeoxyribonucleotides, 6-15 nucleotides long. The degree of hybridization of these oligomers to their RNA complements within the 30S subunit was assessed using nitrocellulose membrane filter binding assays. Specifically, the binding of short DNA oligomers (hexameric and longer) complementary to nucleotides 919-928, 1384-1417, 1490-1505, and 1530-1542 of 16S rRNA was monitored, and in particular how such binding was affected by the change in the activation state of the subunit. We found that nucleotides 1397-1404 comprise an unusually accessible sequence in both active and inactive subunits. Nucleotides 919-924 are partially available for hybridization in active subunits and somewhat more so in inactive subunits. Nucleotides 1534-1542 are freely accessible in active, but only partially accessible in inactive subunits, while nucleotides 1490-1505 and 1530-1533 are inaccessible in both, under the conditions tested. These results are in general agreement with results obtained using other methods and suggest a significant conformational change upon subunit activation.     

Effects of interchain disulfide cross-links on the trypsin cleavage pattern and conformation of myosin subfragment 2

The ability of 5,5'-dithiobis(2-nitrobenzoate) (Nbs2) to produce interchain disulfide cross-links in both the long and short forms of myosin subfragment 2 (S2) and the conformational effects of these cross-links have been investigated. Short S2 (residues 3-287) contains two pairs of Cys residues at positions 66 and 108, and long S2 (residues 1-440) contains an additional pair at position 410. The reaction kinetics of each form of S2 with Nbs2 was biphasic. During the fast kinetic phase the reaction resulted in un-cross-linked species having Nbs-blocked Cys. During the slow phase disulfide-cross-linked species were formed via interchain S-Nbs/SH exchange. For short S2, Cys-66 appeared to react without forming disulfide cross-links, and the Cys- 108 pair reacted with partial cross-linking. For long S2, the Cys-66 pair appeared to react with partial cross-linking, and the Cys pairs at 108 and 410 reacted with complete cross-linking. Mild tryptic digestion of disulfide-cross-linked long S2, under conditions that resulted in partial production of short S2 from un-cross-linked LS2, produced peptides T1a and T1b (residues 1 to approximately 360), with one and two disulfide cross-links, respectively. Further digestion of cross-linked long S2 or cross-linked short S2 resulted in the same shorter fragment, T2, with an NH2-terminus beginning at 103 consistent with a sequence of residues 103-287. Circular dichroism studies on long S2 indicated that the presence of disulfide cross-links changed the thermal unfolding profile of the helix.(ABSTRACT TRUNCATED AT 250 WORDS)     

The structure of a pre-mRNA molecule in solution determined with a site directed cross-linking reagent.

We describe the use of site specific psoralen (SSP) to determine the solution structure of a segment of the human beta globin pre-mRNA. In these experiments, SSP is first delivered as monoadducts to specific nucleotides in the pre-mRNA and subsequently used to form intramolecular RNA-RNA cross-links. The use of this reagent greatly decreases the number of the cross-linked products as compared to generalized psoralen cross-linking. The experiments confirm the locations of previously determined aminomethyltrimethylpsoralen (AMT) cross-links in the human precursor mRNA. In addition, new cross-links consistent with an alternative secondary structure and a small number of cross-links that represent higher order interactions have been determined. Altogether, 42 of 47 cross-links identified in this analysis can be accounted for in a small number of alternative secondary structures and higher order interactions. The site directed cross-linking technique will be useful for the precise determination of RNA secondary and tertiary structures under a variety of experimental conditions.     

Comparison of active and inactive forms of the E. coli 30S ribosomal subunits

Dissociation of E. Coli 70S ribosomes in the presence of 0.1 mM Mg++ yields partially inactivated 30S and 50S subunits. This inactivation can be avoided by dissociating the 70S ribosome in a medium containing 10 mM Mg++. 400 mM Na+. Comparison of the active and inactive forms of the 30S and 50S subunits has led to the following conclusions: 1) The two forms possess identical (50S subunits) or very similar (30S subunits) hydrodynamic properties. No differences in their morphologies is detectable by electron microscopy. 2) They possess the same protein compositions except for the presence of a larger amount of protein S1 in the inactive than in the active form of the 30S subunit. 3) They differ significantly in functional properties: more efficient association of the active than of the inactive forms with the complementary subunit; extensive dimerization of inactive 30S subunits in the presence of 10 mM Mg++; no dimerization of active 30S subunits under the same conditions; six-fold higher peptidyl transferase activity of active as compared to inactive 50S subunits.     

Using a targeted chemical nuclease to elucidate conformational changes in the E. coli 30S ribosomal subunit

Determining the detailed tertiary structure of 16S rRNA within 30S ribosomal subunits remains a challenging problem. The particular structure of the RNA which allows tRNA to effectively interact with the associated mRNA during protein synthesis remains particularly ambiguous. This study utilizes a chemical nuclease, 1, 10-o-phenanthroline-copper, to localize regions of 16S rRNA proximal to the decoding region under conditions in which tRNA does not readily associate with the 30S subunit (inactive conformation), and under conditions which optimize tRNA binding (active conformation). By covalently attaching 1,10-phenanthroline-copper to a DNA oligomer complementary to nucleotides in the decoding region (1396-1403), we have determined that nucleotides 923-929, 1391-1396, and 1190-1192 are within approximately 15 A of the nucleotide base-paired to nucleotide 1403 in inactive subunits, but in active subunits only cleavages (1404-1405) immediately proximal to the 5' end of the hybridized probe remain. These results provide evidence for dynamic movement in the 30S ribosomal subunit, reported for the first time using a targeted chemical nuclease.     

The N-terminal lobes of both regulatory light chains interact with the tail domain in the 10 S-inhibited conformation of smooth muscle myosin

In the presence of ATP, unphosphorylated smooth muscle myosin can form a catalytically inactive monomer that sediments at 10 Svedbergs (10 S). The tail of 10 S bends into thirds and interacts with the regulatory domain. ADP-P(i) is "trapped" at the active site, and consequently the ATPase activity is extremely low. We are interested in the structural basis for maintenance of this off state. Our prior photocross-linking work with 10 S showed that tail residues 1554-1583 are proximal to position 108 in the C-terminal lobe of one of the two regulatory light chains ( Olney, J. J., Sellers, J. R., and Cremo, C. R. (1996) J. Biol. Chem. 271, 20375-20384 ). These data suggested that the tail interacts with only one of the two regulatory light chains. Here we present data, using a photocross-linker on position 59 on the N-terminal lobe of the regulatory light chain (RLC), demonstrating that both regulatory light chains of a single molecule can cross-link to the light meromyosin portion of the tail. Mass spectrometric data show four specific cross-linked regions spanning residues 1428-1571 in the light meromyosin portion of the tail, consistent with cross-linking two RLC to one light meromyosin. In addition, we find that position 59 can cross-link internally to residues 42-45 within the same RLC subunit. The internal cross-link only forms in 10 S and not in unphosphorylated heavy meromyosin (lacking the light meromyosin), suggesting a structural rearrangement within the RLC attributed to the interaction of the tail with the head.     

Photoreaction of psoralen and other furocoumarins with nucleic acids

The 360-nm photoinitiated reactions of certain furo[3,2-g]coumarins with DNA have been examined using ethidium fluorescence assays. Psoralent at 1.85 × 10^?4M gives 3.3 × 10^?5, 1.8 × 10^?5, and 4.5 × 10^?6 interstrand cross-links/nucleotide with DNAs of (A + T) content 70, 60, and 50%, respectively. The relative rates of cross-linking of λ-DNA are 4-methylpsoralen > psoralen > angelicin ? 4-phenylpsoralen. Angelicin (isopsoralen) gives a small (12–14%) but reproducible amount of DNA interstrand cross-links. Addition of netropsin, an antibiotic that binds preferentially to (A + T)-rich regions, to Clostridium perfringens DNA reduces the extent of cross-linking by psoralen from 66 to 10% in 50 min. In contrast, pretreatment of DNA with olivomycin or chromomycin A₃ [which bind to (G + C)-rich regions] has little effect on psoralen cross-linking. Relative rates of monoadduction of furocoumarins to PM2-CCC-DNA detected by thermal depyrimidation and alkaline strand scission is angelicin > 4-methyl-4′,5′-dihydropsoralen > 4′,5′-dihydropsoralen > 3,4-dihydropsoralen (no monoadduction), indicating angelicin is suitable for photolabeling of chromatin. Binding of netropsin to the PM2-DNA prevents cross-linking by angelicin but permits and enhances monoadduction. In contrast neither olivomycin nor chromomycin affects the reaction of angelicin with DNA. In the frozen solution, where the photoinduced cross-linking of DNA by psoralen may be suppressed, psoralen forms monoadducts about twice as readily as angelicin. Subsequent 360-nm irradiation of the psoralen monoadducts at ambient temperatures (and in separate experiments after dialysis to remove unreacted psoralen) completes the cross-links.     

Investigation of the tertiary folding of Escherichia coli ribosomal RNA by intra-RNA cross-linking in vivo

"In vivo" cross-links were introduced into ribosomal RNA by direct ultraviolet irradiation of intact Escherichia coli cells, during growth in a 32P-labelled medium. Ribosomes were isolated from the irradiated cultures, dissociated into subunits and subjected to partial digestion with cobra venom nuclease. The intra-RNA cross-linked fragments were separated by two-dimensional gel electrophoresis and the sites of cross-linking determined, using our published methodology. A comparison with the data previously obtained by this procedure, after irradiation of isolated 30 S and 50 S subunits, showed that in the case of the 50 S subunit nine out of the ten previous cross-links in the 23 S RNA could be identified in the "in vivo" experiments, and correspondingly in the 30 S subunit five out of the six previous cross-links in the 16 S RNA were identified. Some new cross-links were found, as well as two cross-links in the 16 S RNA, which had hitherto only been observed after partial digestion of irradiated 30 S subunits with ribonuclease T1. The relevance of these data to the tertiary folding of the rRNA in situ is discussed, with particular reference to the work of other authors, in which "naked" RNA was used as the substrate for cross-linking and model-building studies.     

Unique cross-link and monoadduct repair characteristics of a xeroderma pigmentosum revertant cell line

Monoadducts and cross-links formed in DNA of human cells by a psoralen derivative, 4'-hydroxy-methyl-4,5',8-trimethylpsoralen (HMT), have been measured by a new, simple method, based on S1 nuclease digestion of 3H-labeled adducts in DNA, that provides rapid information on the repair of both classes of lesions. Normal human fibroblasts and cells from patients with dyskeratosis congenita and xeroderma pigmentosum (XP) group C were capable of removing both monoadducts and cross-links, whereas XP groups A and D failed to remove either. An XP revertant, isolated from a group A cell line on the basis of an acquired mutagen-induced resistance to ultraviolet light, has the unique property of being capable of removing cross-links but not monoadducts. Consistent with this property, the XP revertant was found to be resistant to cell killing by the cross-linking psoralen derivative, HMT, but as sensitive as its parental cell line to a monofunctional psoralen derivative, 5-methylisopsoralen.     

Conformation of 4.5S RNA in the signal recognition particle and on the 30S ribosomal subunit

The signal recognition particle (SRP) from Escherichia coli consists of 4.5S RNA and protein Ffh. It is essential for targeting ribosomes that are translating integral membrane proteins to the translocation pore in the plasma membrane. Independently of Ffh, 4.5S RNA also interacts with elongation factor G (EF-G) and the 30S ribosomal subunit. Here we use a cross-linking approach to probe the conformation of 4.5S RNA in SRP and in the complex with the 30S ribosomal subunit and to map the binding site. The UV-activatable cross-linker p-azidophenacyl bromide (AzP) was attached to positions 1, 21, and 54 of wild-type or modified 4.5S RNA. In SRP, cross-links to Ffh were formed from AzP in all three positions in 4.5S RNA, indicating a strongly bent conformation in which the 5' end (position 1) and the tetraloop region (including position 54) of the molecule are close to one another and to Ffh. In ribosomal complexes of 4.5S RNA, AzP in both positions 1 and 54 formed cross-links to the 30S ribosomal subunit, independently of the presence of Ffh. The major cross-linking target on the ribosome was protein S7; minor cross-links were formed to S2, S18, and S21. There were no cross-links from 4.5S RNA to the 50S subunit, where the primary binding site of SRP is located close to the peptide exit. The functional role of 4.5S RNA binding to the 30S subunit is unclear, as the RNA had no effect on translation or tRNA translocation on the ribosome.     

DNA-protein cross-linking by chromium salts

DNA-protein cross-links were detected in several types of mammalian cells in culture when they were exposed to chromate salts. The cell types included human bronchial epithelial cells — the apparent cell type of origin of the malignancies reported in chromate workers. The level of cross-linking was proportional to the concentration of chromate used. These cross-links appeared to be persistent since no removal was seen after 12 h of repair incubation. A low level of DNA single strand breaks (SSB) were also induced after exposure of the cells to chromate but were rejoined after 4 h of repair incubation. The active form of chromium appears to be the trivalent since chromic but not chromate salts induced DNA-protein cross-links in isolated nuclei. Chromic salts also produced cross-linking between DNA and protein in solution while the hexavalent form was inactive. These data imply that chromate crosses the cell membrane, is reduced to the trivalent form and induces stable linkages of DNA to protein.     

Structure-function correlations (and discrepancies) in the 16S ribosomal RNA from Escherichia coli.

The published model for the three-dimensional arrangement of E coli 16S RNA is re-examined in the light of new experimental information, in particular cross-linking data with functional ligands and cross-links between the 16S and 23S RNA molecules. A growing body of evidence suggests that helix 18 (residues 500-545), helix 34 (residues 1046-1067/1189-1211) and helix 44 (residues 1409-1491) are incorrectly located in the model. It now appears that the functional sites in helices 18 and 34 may be close to the decoding site of the 30S subunit, rather than being on the opposite side of the 'head' of the subunit, as hitherto supposed. Helix 44 is now clearly located at the interface between the 30S and 50S subunits. Furthermore, almost all of the modified bases in both 16S and 23S RNA appear to form a tight cluster near to the upper end of this helix, surrounding the decoding site.     

Comparative cross-linking study on the 50S ribosomal subunit from Escherichia coli

We have carried out an extensive protein-protein cross-linking study on the 50S ribosomal subunit of Escherichia coli using four different cross-linking reagents of varying length and specificity. For the unambiguous identification of the members of the cross-linked protein complexes, immunoblotting techniques using antisera specific for each individual ribosomal protein have been used, and for each cross-link, the cross-linking yield has been determined. With the smallest cross-linking reagent diepoxybutane (4 A), four cross-links have been identified, namely, L3-L19, L10-L11, L13-L21, and L14-L19. With the sulfhydryl-specific cross-linking reagent o-phenylenedimaleimide (5.2 A) and p-phenylenedimaleimide (12 A), the cross-links L2-L9, L3-L13, L3-L19, L9-L28, L13-L20, L14-L19, L16-L27, L17-L32, and L20-L21 were formed; in addition, the cross-link L23-L29 was exclusively found with the shorter o-phenylenedimaleimide. The cross-links obtained with dithiobis(succinimidyl propionate) (12 A) were L1-L33, L2-L9, L2-L9-L28, L3-L19, L9-L28, L13-L21, L14-L19, L16-L27, L17-L32, L19-L25, L20-L21, and L23-L34. The good agreement of the cross-links obtained with the different cross-linking reagents used in this study demonstrates the reliability of our cross-linking approach. Incorporation of our cross-linking results into the three-dimensional model of the 50S ribosomal subunit derived from immunoelectron microscopy yields the locations for 29 of the 33 proteins within the larger ribosomal subunit.     

Selective isolation and detailed analysis of intra-RNA cross-links induced in the large ribosomal subunit of E. coli: a model for the tertiary structure of the tRNA binding domain in 23S RNA.

Intramolecular RNA cross-links were induced within the large ribosomal subunit of E. coli by mild ultraviolet irradiation. Regions of the 23S RNA previously implicated in interactions with ribosomal-bound tRNA were then specifically excised by addressed cleavage using ribonuclease H, in conjunction with synthetic complementary decadeoxyribonucleotides. Individual cross-linked fragments within these regions released by such 'directed digests' were isolated by two-dimensional gel electrophoresis and the sites involved in the cross-links determined using classical oligonucleotide analysis techniques. Using this approach, seven 'new' cross-links could be precisely localised, between positions 1782 and 2608-2609, 1940 and 2554, 1941-1942 and 1964-1965, 1955 and 2552-2553, 2145-2146 and 2202, 2518-2519 and 2544-2545, and between positions 2790-2791 and 2892-2895 in the 23S RNA sequence. These data, in conjunction with data from RNA-protein cross-linking studies carried out in our laboratory, were used to define a model for the tertiary organisation of the tRNA binding domain of 23S RNA 'in situ', in which the specific nucleotides associated with tRNA binding in the 'A' and 'P' sites are clustered at the base of the 'central protuberance' of the 50S subunit.     

N-ethyl maleimide as a probe for the study of functional sites and conformations of 30 S ribosomal subunits

Escherichia coli 30 S ribosomal subunits are inactive in a number of specific functions when Mg²⁺ concentration is reduced to 1 mM, and activity is recovered on heating under appropriate ionic conditions. When active and inactive forms were treated with N-ethyl maleimide, both forms reacted to a similar extent, but the reagent attached mostly to different proteins. Moreover, it caused irreversible inactivation only when reacting with the inactive form of the subunit. Though the activating treatment failed to restore activity to these subunits it did expose the same sulfhydryl groups as are available in the active state for reaction with the maleimide.Different ribosomal activities were eliminated at different maleimide concentrations, permitting the assignment of specific functions to sulfhydryl groups of specific ribosomal proteins. Protein S18 appears to be involved in subunit association, binding of fMet-tRNA and of aminoacyl-tRNA to the P-site. Proteins S1, S14 and S21 are all or in part involved in the binding of aminoacyl-tRNA to the A-site and in the binding of the antibiotic dihydrostreptomycin.The reaction with N-ethyl maleimide thus provides a criterion other than biological activity for characterizing different ribosomal forms and a tool for mapping the 30 S subunit for specific functional sites.     

The epsilon subunit of the F(1)F(0) complex of Escherichia coli. cross-linking studies show the same structure in situ as when isolated.

Four double mutants in the epsilon subunit were generated, each containing two cysteines, which, based on the NMR structure of this subunit, should form internal disulfide bonds. Two of these were designed to generate interdomain cross-links that lock the C-terminal alpha-helical domain against the beta-sandwich (epsilonM49C/A126C and epsilonF61C/V130C). The second set should give cross-linking between the two C-terminal alpha-helices (epsilonA94C/L128C and epsilonA101C/L121C). All four mutants cross-linked with 90-100% efficiency upon CuCl(2) treatment in isolated Escherichia coli ATP synthase. This shows that the structure obtained for isolated epsilon is essentially the same as in the assembled complex. Functional studies revealed increased ATP hydrolysis after cross-linking between the two domains of the subunit but not after cross-linking between the C-terminal alpha-helices. None of the cross-links had any effect on proton pumping-coupled ATP hydrolysis, on DCCD sensitivity of this activity, or on ATP synthesis rates. Therefore, big conformational changes within epsilon can be ruled out as a part of the enzyme function. Protease digestion studies, however, showed that subtle changes do occur, since the epsilon subunit could be locked in an ADP or 5'-adenylyl-beta,gamma-imidodiphosphate conformation by the cross-linking with resulting differences in cleavage rates.