Complementary addressed alkylation of 16S rRNA of Escherichia coli by 2',3'-O-[4-N-methyl-N-(2-chloroethyl)-amino]benzylidene derivatives of oligodeoxyribonucleotides. V. Study of the factors affecting selectivity of modification]

We studied the effect of different factors (reagent concentration, temperature, presence of oligonucleotide-effector (3',5'-diphenazinium derivative of oligodeoxyribonucleotide) stabilizing duplex RNA.reagent) on the selectivity of the site-directed modification of 16S rRNA with 2,3'-O-[4-N-methyl-N-(2-chloroethyl)-amino]-benzylidene derivative of oligonucleotide p(dTTTGCTCCCC)rA (reagent I) under conditions of secondary structure stability. The constant of cooperative binding of the reagent and oligonucleotide-effector with 16s rRNA was determined. The temperature rise from 20 to 40 degrees C brought about a 1.5-fold increase in the relative extent of modification at the target site 771-781. In the presence of oligonucleotide-effector, which is a full complementary copy of the 782-789 fragment of 16S rRNA (reagent concentration is 1 x 10(-6) M), the selectivity of the RNA modification at the target site is doubled and a high level of the modification is retained. When the reagent concentration in the reaction mixture was decreased down to 1 x 10(-7) M, the same level of selectivity was achieved without the oligonucleotide-effector. Under these conditions, however, a drastic (20-fold) drop of the level of the 16S rRNA alkylation was observed.     

The use of RNAse H for digestion of alkylated 16S rRNA at the binding sites of addressed reagents--oligodeoxyribonucleotide derivatives

It is demonstrated that 16S rRNA, complementary-addressed labelled with 2',3'-O-[4-N-methyl-N-(2-chloroethyl)-amino]benzylidene derivatives of oligonucleotides d(pACCTTGTT)rA and d(pTTTGCTCCCC)rA, can be cleaved by RNase H within the adducts, resulted from the modification. Comparative study of the 16S rRNA cleavage with RNase H within the above--mentioned covalent adducts, on the one hand, and within heteroduplexes with the same oligodeoxyribonucleotides, on the other, showed that(i) the complementary-addressed modification proceeds both in perfect and non-per ect complexes; (ii) 16S rRNA is cleaved by RNase H within both perfect and non-perfect complexes resulted from the alkylation, non-perfect complexes being considerably stabilized by the covalent bond between the reagent and the RNA; (iii) non-perfect complexes of 16S rRNA with the free oligodeoxyribonucleotides are unstable even at the high oligonucleotide concentration, so that no cleavage of 16S rRNA in such duplexes is observed. The approach based on cleavage of RNA within covalent adducts resulted from the complementary-addressed RNA modification may be used for fragmentation of RNA molecule in the addressed reagent's binding site.     

Complementary addressed alkylation of Escherichia coli 16S rRNA with 2',3'-O-[4-N-methyl-N-(2-chloroethyl)aminobenzylidene]m derivatives of oligodeoxyribonucleotides. IV. Determination of binding sites of 16S rRNA with benzylidene derivatives of d(pACCTTGTT)rA, d(pTTACGACT)rU, d(TTTGCTCCCC)rA

By site-directed alkylation of 16S rRNA with benzylidene derivatives of d(pACCTTGTT)rA (II), d(pTTACGACT)rU (III), d(pTTTGCTCCCC)rA (IV) (reagents (II)--(IV] followed by the RNase H treatment a number of 16S rRNA fragments have been obtained. Hybridisation of these fragments with restriction fragments of plasmid pKK 3535, containing operon rrnB of E. coli rRNAs, led to the identification of all reagents' binding sites in 16S rRNA. Good correlation is found between estimated stability of non-perfect 16S rRNA.oligodeoxyribonucleotide duplexes and the level of modification of this site with alkylating derivative of the same oligodeoxyribonucleotide. With high concentration of the reagents (II)--(IV) ((2-5) x 10(-5) M) the site-directed alkylation proceeds not only at the desired site but also at other sites corresponding to non-perfect duplexes between 16S rRNA and the reagents. It should be noted that the modification mainly occurs in the non-perfect duplexes, carrying mismatched bases at the termini. Influence of the secondary structure of 16S rRNA on the site-directed modification is discussed.     

Complementary addressed alkylation of Escherichia coli 16S rRNA with 2',3'-O-[4-2-chloroethyl)-N-methylamino]benzylidene derivatives of deoxyribooligonucleotides. II. The nature of rapid interaction at 0 degrees C

Fast non-covalent interactions of 16S rRNA Escherichia coli with 14C labeled 2',3'-O-[4-N-(2-chloroethyl)-N-methylamino]benzylidene derivatives of deoxyribooligonucleotides d(pACCTTGTT)rA, d[pTTACGATC)rU, d(pTTTGCTCCCC)rA (less than[14C]CHRCl-reagents) observed at 0 degrees C were investigated. It was shown, that 16S rRNA and [14C]CHRCl-reagents at 0 degrees C form stable complexes which can not be disrupted under mild acidic conditions (pH 4, 40 degrees C) and under denaturing conditions (7 M urea, 50 degrees C), but are completely disrupted in the course of centrifugation in sucrose density gradient in the presence of SDS. Formation of such complexes of 16S rRNA with greater than[14C]CHRCl-reagents at 0 degrees C was observed due to the presence in the reagent preparation of a number of unidentified products, formed in the course of the synthesis of benzylidene derivatives, and having a hydrophobicity larger, than those for greater than CHRCl-derivatives of deoxyribooligonucleotide. Preparation of [14C]CHRCl-reagents, subjected for purification by reverse-phase chromatography, were unable to form such a complex with 16S rRNA at 0 degrees C. Studies on the complementary addressed modification at 0 degrees C (or incubation at 0 degrees C) with the use of the oligonucleotide benzylidene derivatives not purified from hydrophobic contaminations may lead to alkylation within these complexes during subsequent treatments and in such a way give incorrect information about the level of alkylation within the complex under investigation.     

Methyltransferase that modifies guanine 966 of the 16 S rRNA: functional identification and tertiary structure

N(2)-Methylguanine 966 is located in the loop of Escherichia coli 16 S rRNA helix 31, forming a part of the P-site tRNA-binding pocket. We found yhhF to be a gene encoding for m(2)G966 specific 16 S rRNA methyltransferase. Disruption of the yhhF gene by kanamycin resistance marker leads to a loss of modification at G966. The modification could be rescued by expression of recombinant protein from the plasmid carrying the yhhF gene. Moreover, purified m(2)G966 methyltransferase, in the presence of S-adenosylomethionine (AdoMet), is able to methylate 30 S ribosomal subunits that were purified from yhhF knock-out strain in vitro. The methylation is specific for G966 base of the 16 S rRNA. The m(2)G966 methyltransferase was crystallized, and its structure has been determined and refined to 2.05A(.) The structure closely resembles RsmC rRNA methyltransferase, specific for m(2)G1207 of the 16 S rRNA. Structural comparisons and analysis of the enzyme active site suggest modes for binding AdoMet and rRNA to m(2)G966 methyltransferase. Based on the experimental data and current nomenclature the protein expressed from the yhhF gene was renamed to RsmD. A model for interaction of RsmD with ribosome has been proposed.     

Specific fragmentation of tRNA and rRNA at a 7-methylguanine residue in the presence of methylated carrier RNA

The reaction of site-specific cleavage of tRNA at a 7-methylguanine residue, including subsequent treatment with sodium borohydride and aniline [Wintermeyer, W. and Zachau, H.G. (1975) FEBS Lett. 58, 306-309], was shown to work only within a certain range of tRNA concentrations (higher than 30 microM). The Escherichia coli 16S rRNA, which contained a unique m7G (position 527), could not be split by this method when taken at any concentration. It was found that the presence of statistically methylated carrier RNA in the reaction mixture at the borohydride stage significantly stimulates site-specific fragmentation of 16S rRNA and 32P-labeled tRNAs. Direct sequencing proved that 16S rRNA and tRNA are cleaved by this procedure successfully at the m7G residue. The E. coli 16S rRNA was preparatively cleaved by the described procedure into two fragments. The 5'-terminal fragment (1-526) and the 3'-terminal fragment (528-1542) were isolated in the pure form and their secondary structure investigated by the circular dichroism method. The results of this study showed that the secondary and tertiary structures of the 5'-terminal one-third of the 16S rRNA are at least as ordered as those of intact 16S rRNA or its 3'-terminal two-thirds.     

The spoU gene of Escherichia coli, the fourth gene of the spoT operon, is essential for tRNA (Gm18) 2'-O-methyltransferase activity.

We have evidence that the open reading frame previously denoted spoU is necessary for tRNA (Gm18) 2'-O-methyltransferase activity. The spoU gene is located in the gmk-rpoZ-spoT-spoU-recG operon at 82 minutes on the Escherichia coli chromosome. The deduced amino acid sequence of spoU shows strong similarities to previously characterized 2'-O-methyltransferases. Comparison of the nucleoside modification pattern of hydrolyzed tRNA, 16S rRNA and 23S rRNA from wild-type and spoU null mutants showed that the modified nucleoside 2'-O-methylguanosine (Gm), present in a subset of E. coli tRNAs at residue 18, is completely absent in the spoU mutant, suggesting that spoU encodes tRNA (Gm18) 2'-O-methyltransferase. Nucleoside modification of 16S and 23S rRNA was unaffected in the spoU mutant. Insertions in the downstream recG gene did not affect RNA modification. Absence of Gm18 in tRNA does not influence growth rate under the tested conditions and does not interfere with activity of the SupF amber suppressor, a suppressor tRNA that normally has the Gm18 modification. We suggest that the spoU gene be renamed trmH (tRNA methylation).     

Neighborhood of 16S rRNA nucleotides U788/U789 in the 30S ribosomal subunit determined by site-directed crosslinking.

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.     

Subribosomal particle analysis reveals the stages of bacterial ribosome assembly at which rRNA nucleotides are modified

Modified nucleosides of ribosomal RNA are synthesized during ribosome assembly. In bacteria, each modification is made by a specialized enzyme. In vitro studies have shown that some enzymes need the presence of ribosomal proteins while other enzymes can modify only protein-free rRNA. We have analyzed the addition of modified nucleosides to rRNA during ribosome assembly. Accumulation of incompletely assembled ribosomal particles (25S, 35S, and 45S) was induced by chloramphenicol or erythromycin in an exponentially growing Escherichia coli culture. Incompletely assembled ribosomal particles were isolated from drug-treated and free 30S and 50S subunits and mature 70S ribosomes from untreated cells. Nucleosides of 16S and 23S rRNA were prepared and analyzed by reverse-phase, high-performance liquid chromatography (HPLC). Pseudouridines were identified by the chemical modification/primer extension method. Based on the results, the rRNA modifications were divided into three major groups: early, intermediate, and late assembly specific modifications. Seven out of 11 modified nucleosides of 16S rRNA were late assembly specific. In contrast, 16 out of 25 modified nucleosides of 23S rRNA were made during early steps of ribosome assembly. Free subunits of exponentially growing bacteria contain undermodified rRNA, indicating that a specific set of modifications is synthesized during very late steps of ribosome subunit assembly.     

The identification of spermine binding sites in 16S rRNA allows interpretation of the spermine effect on ribosomal 30S subunit functions

A photoreactive analogue of spermine, N¹-azidobenzamidino (ABA)-spermine, was covalently attached after irradiation to Escherichia coli 30S ribosomal subunits or naked 16S rRNA. By means of RNase H digestion and primer extension, the cross-linking sites of ABA-spermine in naked 16S rRNA were characterised and compared with those identified in 30S subunits. The 5′ domain, the internal and terminal loops of helix H24, as well as the upper part of helix H44 in naked 16S rRNA, were found to be preferable binding sites for polyamines. Association of 16S rRNA with ribosomal proteins facilitated its interaction with photoprobe, except for 530 stem–loop nt, whose modification by ABA-spermine was abolished. Association of 30S with 50S subunits, poly(U) and AcPhe-tRNA (complex C) further altered the susceptibility of ABA-spermine cross-linking to 16S rRNA. Complex C, modified in its 30S subunit by ABA-spermine, reacted with puromycin similarly to non-photolabelled complex. On the contrary, poly(U)-programmed 70S ribosomes reconstituted from photolabelled 30S subunits and untreated 50S subunits bound AcPhe-tRNA more efficiently than untreated ribosomes, but were less able to recognise and reject near cognate aminoacyl-tRNA. The above can be interpreted in terms of conformational changes in 16S rRNA, induced by the incorporation of ABA-spermine.     

Organization of the 16S rRNA around its 5' terminus determined by photochemical crosslinking in the 30S ribosomal subunit

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.     

Interaction of the antibiotics clindamycin and lincomycin with Escherichia coli 23S ribosomal RNA.

Interaction of the antibiotics clindamycin and lincomycin with Escherichia coli ribosomes has been compared by chemical footprinting. The protection afforded by both drugs is limited to the peptidyl transferase loop of 23S rRNA. Under conditions of stoichiometric binding at 1 mM drug concentration in vitro, both drugs strongly protect 23S rRNA bases A2058 and A2451 from dimethyl sulphate and G2505 from kethoxal modification; G2061 is also weakly protected from kethoxal. The modification patterns differ in that A2059 is additionally protected by clindamycin but not by lincomycin. The affinity of the two drugs for the ribosome, estimated by footprinting, is approximately the same, giving Kdiss values of 5 microM for lincomycin and 8 microM for clindamycin. The results show that in vitro the drugs are equally potent in blocking their ribosomal target site. Their inhibitory effects on peptide bond formation could, however, be subtly different.     

Secondary structure model for bacterial 16S ribosomal RNA: phylogenetic, enzymatic and chemical evidence.

We have derived a secondary structure model for 16S ribosomal RNA on the basis of comparative sequence analysis, chemical modification studies and nuclease susceptibility data. Nucleotide sequences of the E. coli and B. brevis 16S rRNA chains, and of RNAse T1 oligomer catalogs from 16S rRNAs of over 100 species of eubacteria were used for phylogenetic comparison. Chemical modification of G by glyoxal, A by m-chloroperbenzoic acid and C by bisulfite in naked 16S rRNA, and G by kethoxal in active and inactive 30S ribosomal subunits was taken as an indication of single stranded structure. Further support for the structure was obtained from susceptibility to RNases A and T1. These three approaches are in excellent agreement. The structure contains fifty helical elements organized into four major domains, in which 46 percent of the nucleotides of 16S rRNA are involved in base pairing. Phylogenetic comparison shows that highly conserved sequences are found principally in unpaired regions of the molecule. No knots are created by the structure.     

Recovery of microbial diversity and activity during bioremediation following chemical oxidation of diesel contaminated soils

To improve the coupling of in situ chemical oxidation and in situ bioremediation, a systematic analysis was performed of the effect of chemical oxidation with Fenton's reagent, modified Fenton's reagent, permanganate, or persulfate, on microbial diversity and activity during 8 weeks of incubation in two diesel-contaminated soils (peat and fill). Chemical oxidant and soil type affected the microbial community diversity and biodegradation activity; however, this was only observed following treatment with Fenton's reagent and modified Fenton's reagent, and in the biotic control without oxidation. Differences in the highest overall removal efficiencies of 69 % for peat (biotic control) and 59 % for fill (Fenton's reagent) were partially explained by changes in contaminant soil properties upon oxidation. Molecular analysis of 16S rRNA and alkane monooxygenase (alkB) gene abundances indicated that oxidation with Fenton's reagent and modified Fenton's reagent negatively affected microbial abundance. However, regeneration occurred, and final relative alkB abundances were 1–2 orders of magnitude higher in chemically treated microcosms than in the biotic control. 16S rRNA gene fragment fingerprinting with DGGE and prominent band sequencing illuminated microbial community composition and diversity differences between treatments and identified a variety of phylotypes within Alpha-, Beta-, and Gammaproteobacteria. Understanding microbial community dynamics during coupled chemical oxidation and bioremediation is integral to improved biphasic field application.     

Differential assembly of 16S rRNA domains during 30S subunit formation

Rapid and accurate assembly of the ribosomal subunits, which are responsible for protein synthesis, is required to sustain cell growth. Our best understanding of the interaction of 30S ribosomal subunit components (16S ribosomal RNA [rRNA] and 20 ribosomal proteins [r-proteins]) comes from in vitro work using Escherichia coli ribosomal components. However, detailed information regarding the essential elements involved in the assembly of 30S subunits still remains elusive. Here, we defined a set of rRNA nucleotides that are critical for the assembly of the small ribosomal subunit in E. coli. Using an RNA modification interference approach, we identified 54 nucleotides in 16S rRNA whose modification prevents the formation of a functional small ribosomal subunit. The majority of these nucleotides are located in the head and interdomain junction of the 30S subunit, suggesting that these regions are critical for small subunit assembly. In vivo analysis of specific identified sites, using engineered mutations in 16S rRNA, revealed defective protein synthesis capability, aberrant polysome profiles, and abnormal 16S rRNA processing, indicating the importance of these residues in vivo. These studies reveal that specific segments of 16S rRNA are more critical for small subunit assembly than others, and suggest a hierarchy of importance.     

The RNA N-glycosidase activity of ricin A-chain

The RNA N-glycosidase activity of ricin A-chain has been characterized. When rat liver ribosomes were used as substrates, the A-chain cleaved the N-glycosidic bond at A-4324 in 28S rRNA. An apparent Michaelis constant (Km) for the reaction was determined to be 2.6 microM and the turnover number (Kcat) was 1777 min-1. When naked rRNA was the substrate, the A-chain cleaved the same bond in 28S rRNA but at a greatly reduced rate. The Km value was 5.8 microM. The results suggest that the A-chain has a similar affinity for 28S rRNA in both ribosomes and the naked states. When the deproteinized Escherichia coli rRNA was the substrates, ricin A-chain cleaved a N-glycosidic bond at A-2600 in 23S rRNA which corresponds to the ricin-site in 28S rRNA of rat liver ribosomes, while the A-chain has little activity on 23S rRNA in the ribosomes. The results suggest that ricin A-chain acts directly on RNA by recognizing a certain structure in the molecules. Using the secondary structure models for each species of rRNA, we have deduced a loop and stem structure having GAGA in the loop to be a minimum requirement for the substrate of ricin A-chain.