Substrate specificity and properties of the Escherichia coli 16S rRNA methyltransferase, RsmE

The small ribosome subunit of Escherichia coli contains 10 base-methylated sites distributed in important functional regions. At present, seven enzymes responsible for methylation of eight bases are known, but most of them have not been well characterized. One of these enzymes, RsmE, was recently identified and shown to specifically methylate U1498. Here we describe the enzymatic properties and substrate specificity of RsmE. The enzyme forms dimers in solution and is most active in the presence of 10-15 mM Mg(2+) and 100 mM NH(4)Cl at pH 7-9; however, in the presence of spermidine, Mg(2+) is not required for activity. While small ribosome subunits obtained from an RsmE deletion strain can be methylated by purified RsmE, neither 70S ribosomes nor 50S subunits are active. Likewise, 16S rRNA obtained from the mutant strain, synthetic 16S rRNA, and 3' minor domain RNA are all very poor or inactive as substrates. 30S particles partially depleted of proteins by treatment with high concentrations of LiCl or in vitro reconstituted intermediate particles also show little or no methyl acceptor activity. Based on these data, we conclude that RsmE requires a highly structured ribonucleoprotein particle as a substrate for methylation, and that methylation events in the 3' minor domain of 16S rRNA probably occur late during 30S ribosome assembly.     

Magnesium ion induced proton release as a probe for the polyelectrolytic structure of ribosomal RNAs and subunits

E coli ribosomes and rRNA's released 20 to 50 protons upon jump of magnesium ion concentration from 1 mM to 20 mM. The Mg2+-induced proton release was measured separately for 16S rRNA, 23S rRNA, 30S subunit, and 50S subunit by a new spectrophotometric method that had a much better sensitivity than the pH-stat method. The proton release from the subunits and rRNA's were similar in the number of protons, the pH dependence that had a minimum at neutral pH, and the upward concaveness of the Scatchard plot. From these results, the main source of protons in ribosomal subunits was assigned to nucleotide bases of rRNA's that showed a downward pKa shift upon Mg2+-ion binding. The subunits and rRNA's, however, differed in the proton release. 16S rRNA released protons somewhat more effectively than 23S rRNA, while 30S subunit released protons 2 to 5 times more effectively than 50S subunit. The marked difference between the two subunits suggest that ionizable bases in 16S and 23S rRNA's are covered and their pKa values are shifted by ribosomal proteins to different extents. The association of 30S and 50S subunits induced little proton release, showing that few ionizable groups with pKa near neutral pH are involved in the association. E. coli tRNA and poly U also showed Mg2+-induced proton release. The amounts of protons released from rRNA's, tRNA, and poly U were roughly proportional to the amount of bases not hydrogen bonded. The Mg2+-induced proton release from the natural and synthetic RNA's can be explained by the electrostatic field effect of polyphosphate backbones on bases not hydrogen bonded, as proposed in a previous paper. It also reflects the conformational structure of each RNA molecule.     

Poly(4-thiouridylic acid) as messenger RNA and its application for photoaffinity labelling of the ribosomal mRNA binding site.

Poly(4-thiouridylic acid) [poly(s4U)] synthesized by polymerization of 4-thiouridine 5'-diphosphate with Escherichia coli polynucleotide phosphorylase (EC 2.7.7.8) acts as messenger RNA in vitro in a protein-synthesizing system from E. coli. It stimulates binding of Phe-tRNA to ribosomes both in the presence of EF-Tu-Ts at 5 mM Mg2+ concentration and nonenzymatically at 20 mM Mg2+ concentration. It codes for the synthesis of polyphenylalanine. Poly(s4U) competes with poly(U) for binding to E. coli ribosomes. Light of 330 nm photoactivates poly(s4U) thus making it a useful photoaffinity label for the ribosomal mRNA binding site. Upon irradiation of 70-S ribosomal complexes, photoreaction occurs with ribosomal proteins as well as 16-S RNA. Ribosomes pre-incubated with R17 RNA are protected against the photoaffinity reaction. The labelling of 16-S RNA can be reduced by treatment of ribosomes with colicin E3.     

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.     

Destabilization of the secondary structure of RNA by ribosomal protein S1 from escherichia coli

S1 is an acidic protein associated with the 3′ end of 16S RNA; it is indispensable for ribosomal binding of natural mRNA. We find that S1 unfolds single stranded stacked or helical polynucleotides (poly rA, poly rC, poly rU). It prevents the formation of poly (rA + rU) and poly (rI + rC) duplexes at 10–25 mM NaCl but not at 50–100 mM NaCl. Partial, salt reversible denaturation is also seen with coliphage MS2 RNA, $\text{E. coli}$ rRNA and tRNA. Generally, only duplex structures with a Tm greater than about 55° are formed in the presence of S1. The protein unfolds single stranded DNA but not poly d(A·T).     

Specific polyadenylation and purification of total messenger RNA from Escherichia coli.

Obtaining pure mRNA preparations from prokaryotes has been difficult, if not impossible, for want of a poly(A) tail on these messages. We have used poly(A) polymerase from yeast to effect specific polyadenylation of Escherichia coli polysomal mRNA in the presence of magnesium and manganese. The polyadenylated total mRNA, which could be subsequently purified by binding to and elution from oligo(dT) beads, had a size range of 0.4-4.0 kb. We have used hybridization to a specific plasmid-encoded gene to further confirm that the polyadenylated species represented mRNA. Withdrawal of Mg2+ from the polyadenylation reaction resulted in addition of poly(A) to 16S rRNA despite the presence of Mn2+, indicating the vital role of Mg2+ in maintaining the native structure of polysomes. Complete dissociation of polysomes into ribosomal subunits resulted in quantitative polyadenylation of both 16S and 23S rRNA species. Chromosomal lacZ gene-derived messages were quantitatively recovered in the oligo(dT)-bound fraction, as demonstrated by RT-PCR analysis. Potential advantages that accrue from the availability of pure total mRNA from prokaryotes is discussed.     

Complementarity of sequences in low molecular weight RNAs to regions of messenger and ribosomal RNAs.

Total low molecular weight nuclear RNAs of mouse ascites cells have been labeled in vitro and used as probes to search for complementary sequences contained in nuclear or cytoplasmic RNA. From a subset of hybridizing lmw RNAs, two major species of 58,000 and 35,000 mol. wt. have been identified as mouse 5 and 5.8S ribosomal RNA. Mouse 5 and 5.8S rRNA hybridize not only to 18 and 28S rRNA, respectively, but also to nuclear and cytoplasmic poly(A+) RNA. Northern blot analysis and oligo-dT cellulose chromatography have confirmed the intermolecular base-pairing of these two small rRNA sequences to total poly(A+) RNA as well as to purified rabbit globin mRNA. 5 and 5.8S rRNA also hybridize with positive (coding) but not negative (noncoding) strands of viral RNA. Temperature melting experiments have demonstrated that their hybrid stability with mRNA sequences is comparable to that observed for the 5S:18S and 5.8S:28S hybrids. The functional significance of 5 and 5.8S rRNA base-pairing with mRNAs and larger rRNAs is unknown, but these interactions could play important coordinating roles in ribosome structure, subunit interaction, and mRNA binding during translation.     

Covalent cross-linking of transfer ribonucleic acid to the ribosomal P site. Mechanism and site of reaction in transfer ribonucleic acid.

The covalent cross-linking of unmodified Escherichia coli N-acetylvalyl-tRNA to the 16S RNA of Escherichia coli ribosomes upon near-UV irradiation previously reported by us [Schwartz, I., & Ofengand, J. (1978) Biochemistry 17, 2524--2530] has been studied further. Up to 70% of the unmodified tRNA, nonenzymatically bound to tight-couple ribosomes at 7 mM Mg2+, could be cross-linked by 310--335-nm light. Covalent attachment was solely to the 16S RNA. It was dependent upon both irradiation and the presence of mRNA but was unaffected by the presence or absence of 4-thiouridine in the tRNA. The kinetics of cross-linking showed single-hit behavior. Twofold more cross-linking was obtained w-th tight-couple ribosomes than with salt-washed particles. Puromycin treatment after irradiation released the bound N-acetyl[3H]valine, demonstrating that the tRNA was covalently bound at the P site and that irradiation and covalent linking did not affect the peptidyl transferase reaction. Cross-linking was unaffected by the presence of O2, argon, ascorbate (1 mM), or mercaptoethanol (10 mM). Prephotolysis of a mixture of tRNA and ribosomes in the absence of puly(U2,G) did not block subsequent cross-linking in its presence nor did it generate any long-lived chemically reactive species. There was a strong tRNA specificity. E. coli tRNA1Val and tRNA1Ser and Bacillus subtilis tRNAVal and tRNAThr could be cross-linked, but E. coli tRNA2Val, 5-fluorouracil-substituted tRNA1Val, tRNAPhe, or tRNAFMet could not. By sequence comparison of the reactive and nonreactive tRNAs, the site of attachment in the tRNA was deduced to be the 5'-anticodon base, cmo5U, or ,o5U in all of the reactive tRNAs. The attachment site in 16S RNA is described in the accompanying paper [Zimmerman, R. A., Gates, S. M., Schwartz, I., & Ofengand, J. (1979) Biochemistry (following paper in this issue)]. The link between tRNA and 16S RNA is either direct or involves mRNA bases at most two nucleotides apart since use of the trinucleotide GpUpU in place of poly(U2,G) to direct the binding and cross-linking of N-acetylvalyl-tRNA to the P site did not affect either the rate or yield of cross-linking. Both B. subtilis tRNAVal (mo5U) and E. coli tRNA1Val (cmo5U) gave the same rate and yield of cross-linking when directed by the trinucleotide GpUpU. Therefore, the presence of the charged carboxyl group in the cmo5U-containing tRNA apparently does not markedly perturb the orientation of this base with respect to its reaction partner in the 16S RNA. The cross-linking of AcVal-tRNA only takes place from the P site. At 75 mM KCl and 75 mM NH4Cl, less than 0.4% cross-linking was found at the A site, while 55.5% was obtained at the P site. However, when the salt concentration was lowered to 50 mM NH4Cl, 5% cross-linking to the A site was detected, compared to 49% at the P site. Thus, a simple change in the ionic strength of the incubation mixture was able to alter the affinity labeling pattern of the ribosome.     

5'-Terminal cap structures of oligo(uridylic acid)-containing messenger ribonucleic acid from HeLa cells: comparison with other ribonucleic acid subpopulations

The 5'-terminal cap structures of 32P-labeled oligo(uridylic acid)-containing messenger ribonucleic acid [oligo(U+)mRNA] isolated from HeLa cell polyadenylated [poly(A+)] mRNA were analyzed and compared to those of the poly(A+) mRNA. A method employing P1 nuclease, alkaline phosphatase, and adsorption to activated charcoal showed that the types of cap core (m7 GpppXm) in oligo(U+) mRNA were essentially identical with those in poly(A+) mRNA. Analysis of RNase T2 digestion products of oligo(U+) mRNA demonstrated the presence of both cap 1 (m7GpppXmpYp) and cap 2 (m7GpppXmpYmpZp) in this subpopulation, confirming its cytoplasmic location. The base compositions of these two types of caps were different from each other and nonrandom but did not differ significantly between oligo(U+) and poly(A+) m RNA. The only observed difference between the mRNA populations was a higher ratio of cap 1 and cap 2 in the former. Possible implications of these findings for the relationship between oligo(U+) mRNA and poly(A+) mRNA are discussed.     

RNA-RNA and RNA-protein interactions in 30 S ribosomal subunits. Association of 16 S rRNA fragments in the presence of ribosomal proteins.

The E. coli 16 S rRNA with single-site breaks centered at position 777 or 785 was obtained by RNase H site-specific cleavage of rRNA. Spontaneous dissociation of the cleaved 16 S rRNA into fragments occurred under 'native' conditions. The reassociation of the 16 S rRNA fragments was possible only in the presence of ribosomal proteins. The combination of S4 and S16(S17) ribosomal proteins interacting mainly with the 5'-end domain of 16 S rRNA was sufficient for reassociation of the fragments. The 30 S subunits with fragmented RNA at ca. 777 region retained some poly(U)-directed protein synthetic activity.     

Diffusely bound Mg2+ ions slightly reorient stems I and II of the hammerhead ribozyme to increase the probability of formation of the catalytic core

The hammerhead ribozyme is one of the best-studied small RNA enzymes, yet is mechanistically still poorly understood. We measured the Mg(2+) dependencies of folding and catalysis for two distinct hammerhead ribozymes, HHL and HH alpha. HHL has three long helical stems and was previously used to characterize Mg(2+)-induced folding. HH alpha has shorter stems and an A.U tandem next to the cleavage site that increases activity approximately 10-fold at 10 mM Mg(2+). We find that both ribozymes cleave with fast rates (5-10 min(-1), at pH 8 and 25 degrees C) at nonphysiologically high Mg(2+) concentrations, but with distinct Mg(2+) dissociation constants for catalysis: 90 mM for HHL and 10 mM for HH alpha. Using time-resolved fluorescence resonance energy transfer, we measured the stem I-stem II distance distribution as a function of Mg(2+) concentration, in the presence and absence of 100 mM Na(+), at 4 and 25 degrees C. Our data show two structural transitions. The larger transition (with Mg(2+) dissociation constants in the physiological range of approximately 1 mM, below the catalytic dissociation constants) brings stems I and II close together and is hindered by Na(+). The second, globally minor, rearrangement coincides with catalytic activation and is not hindered by Na(+). Additionally, the more active HH alpha exhibits a higher flexibility than HHL under all conditions. Finally, both ribozyme-product complexes have a bimodal stem I-stem II distance distribution, suggesting a fast equilibrium between distinct conformers. We propose that the role of diffusely bound Mg(2+) is to increase the probability of formation of a properly aligned catalytic core, thus compensating for the absence of naturally occurring kissing-loop interactions.     

A synthetic alanyl-initiator tRNA with initiator tRNA properties as determined by fluorescence measurements: comparison to a synthetic alanyl-elongator tRNA.

Two synthetic tRNAs have been generated that can be enzymatically aminoacylated with alanine and have AAA anticodons to recognize a poly(U) template. One of the tRNAs (tRNA(eAla/AAA)) is nearly identical to Escherichia coli elongator tRNA(Ala). The other has a sequence similar to Escherichia coli initiator tRNA(Met) (tRNA(iAla/AAA)). Although both tRNAs can be used in poly(U)-directed nonenzymatic initiation at 15 mM Mg2+, only the elongator tRNA can serve for peptide elongation and polyalanine synthesis. Only the initiator tRNA can be bound to 30S ribosomal subunits or 70S ribosomes in the presence of initiation factor 2 (IF-2) and low Mg2+ suggesting that it can function in enzymatic peptide initiation. A derivative of coumarin was covalently attached to the alpha amino group of alanine of these two Ala-tRNA species. The fluorescence spectra, quantum yield and anisotropy for the two Ala-tRNA derivatives are different when they are bound to 70S ribosomes (nonenzymatically in the presence of 15 mM Mg2+) indicating that the local environment of the probe is different. Also, the effect of erythromycin on their fluorescence is quite different, suggesting that the probes and presumably the alanine moiety to which they are covalently linked are in different positions on the ribosomes.     

RNA isolation and fractionation with compaction agents

A new approach to the isolation of RNA from bacterial lysates employs selective precipitation by compaction agents, such as hexammine cobalt and spermidine. Using 3.5 mM hexammine cobalt, total RNA can be selectively precipitated from a cell lysate. At a concentration of 2 mM hexammine cobalt, rRNA can be fractionated from low molecular weight RNA. The resulting RNA mixture is readily resolved to pure 5S and mixed 16S/23S rRNA by nondenaturing anion-exchange chromatography. Using a second stage of precipitation at 8 mM hexammine cobalt, the low molecular weight RNA fraction can be isolated by precipitation. Compaction precipitation was also applied to the purification of an artificial stable RNA derived from Escherichia coli 5S rRNA and to the isolation of an Escherichia coli-expressed ribozyme.     

A decreased aminoacyl-transfer-ribonucleic acid-binding capacity of 40S ribosomal subunits resulting from hypophysectomy of the rat

A technique that permitted the reversible dissociation of rat liver ribosomes was used to study the difference in protein-synthetic activity between liver ribosomes of normal and hypophysectomized rats. Ribosomal subunits of sedimentation coefficients 38S and 58S were produced from ferritin-free ribosomes by treatment with 0.8m-KCl at 30 degrees C. These recombined to give 76S monomers, which were as active as untreated ribosomes in incorporating phenylalanine in the presence of poly(U). Subunits from normal and hypophysectomized rats were recombined in all possible combinations and the ability of the hybrid ribosomes to catalyse polyphenylalanine synthesis was measured. The results show that the defect in ribosomes of hypophysectomized rats lies only in the small ribosomal subunit. The 40S but not the 60S subunit of rat liver ribosomes bound poly(U). The only requirement for the reaction was Mg(2+), the optimum concentration of which was 5mm. No apparent difference was seen between the poly(U)-binding abilities of 40S ribosomal subunits from normal or hypophysectomized rats. Phenylalanyl-tRNA was bound by 40S ribosomal subunits in the presence of poly(U) by either enzymic or non-enzymic reactions. Non-enzymic binding required a Mg(2+) concentration in excess of 5mm and increased linearly with increasing Mg(2+) concentrations up to 20mm. At a Mg(2+) concentration of 5mm, GTP and either a 40-70%-saturated-(NH(4))(2)SO(4) fraction of pH5.2 supernatant or partially purified aminotransferase I was necessary for binding of aminoacyl-tRNA. Hypophysectomy of rats resulted in a decreased binding of aminoacyl-tRNA by 40S ribosomal subunits.     

Two distinct poly(A) polymerases isolated from the cytoplasm of Ehrlich ascites tumour cells

The poly(A) polymerases from the cytosol and ribosomal fractions of Ehrlich ascites tumour cells are isolated and partially purified by DEAE-cellulose and phosphocellulose column chromatography. Two distinct enzymes are identified: (a) a cytosol Mn2+-dependent poly(A) polymerase (ATP:RNA adenylyltransferase) and (b) a ribosome-associated enzyme defined tentatively as ATP(UTP): RNA nucleotidyltransferase. The cytosol poly(A) polymerase is strictly Mn2+-dependent (optimum at 1 mM Mn2+) and uses only ATP as substrate, poly(A) is a better primer than ribosomal RNA. The purified enzyme is free of poly(A) hydrolase activity, but degradation of [3H]poly(A) takes place in the presence of inorganic pyrophosphate. Most likely this enzyme is of nuclear origin. The ribosomal enzyme is associated with the ribosomes but it is found also in free state in the cytosol. The purified enzyme uses both ATP and UTP as substrates. The substrate specificity varies depending on ionic conditions: the optimal enzyme activity with ATP as substrate is at 1 mM Mn2+, while that with UTP as substrate is at 10--20 mM Mg2+. The enzymes uses both ribosomal RNA and poly(A) [but not poly(U)] as primers. The purified enzyme is free of poly(A) hydrolase activity.     

Purification and characterization of an endoribonuclease from nucleoplasm and nucleoli of HeLa cells.

An endoribonuclease has been isolated from HeLa cell nuclei. Approximately 70% of the enzyme appears to be nucleolar bound; 30% is in the nucleoplasm. Studies of the purified enzyme reveal that the enzyme is an endonuclease of estimated molecular weight 16,000. It produces oligonucleotides bearing 5'-phosphate end groups. The enzyme degrades poly(C) and poly(U), as well as rRNA and heterogeneous nuclear RNA, Poly(A), double-stranded RNA, and DNA are not cleaved. The enzyme is heat-labile and is inhibited by 10mM Mg2+ and 50 mM NaCl. The enzyme is probably distinct from previously described nuclear endonucleases.     

Association of poly(adenylate)-deficient messenger ribonucleic acid with membranes in mouse kidney

To describe further the metabolism of messenger ribonucleic acid (mRNA) in mouse kidney, we examined newly synthesized mRNA deficient in poly(adenylate) [poly(A)]. Approximately 50% of renal polysomal mRNA that labeled selectively in the presence of the pyrimidine analogue 5-fluoroorotic acid lacks or is deficient in poly(A) as defined by its ability to bind to poly(A) affinity columns. Nearly one-half of this poly(A)-deficient mRNA is associated uniquely with a cellular membrane fraction detected by sedimentation of renal cytoplasm in sucrose density gradients containing EDTA and nonionic detergents. Poly(A+) mRNA and poly(A)-deficient mRNA [poly(A-) mRNA] have similar modal sedimentation coefficients (20-22 S) and similar cytoplasmic distribution. Although 95% of newly synthesized poly(A+) mRNA is released in 10 mM EDTA as 20-90 S ribonucleoproteins from polysomes greater than 80 S, only 55% of poly(A)-deficient mRNA is released under the same conditions. Poly(A)-deficient mRNA recovered from greater than 80 S ribonucleoproteins resistant to EDTA treatment lacks ribosomal RNA, is similar in size to poly(A+) mRNA, and is associated with membranous structures, since 70% of poly(A)-deficient mRNA in EDTA-resistant ribonucleoproteins is released into the 20-80 S region by solubilizing membranes with 1% Triton X-100. These membrane-associated renal poly(A-) mRNAs could have unique coding or regulatory functions.