Ribosomal RNA modification enzymes stimulate large ribosome subunit assembly in E. coli.

Ribosomal RNA modifications are introduced by specific enzymes during ribosome assembly in bacteria. Deletion of individual modification enzymes has a minor effect on bacterial growth, ribosome biogenesis, and translation, which has complicated the definition of the function of the enzymes and their products. We have constructed an Escherichia coli strain lacking 10 genes encoding enzymes that modify 23S rRNA around the peptidyl-transferase center. This strain exhibits severely compromised growth and ribosome assembly, especially at lower temperatures. Re-introduction of the individual modification enzymes allows for the definition of their functions. The results demonstrate that in addition to previously known RlmE, also RlmB, RlmKL, RlmN and RluC facilitate large ribosome subunit assembly. RlmB and RlmKL have functions in ribosome assembly independent of their modification activities. While the assembly stage specificity of rRNA modification enzymes is well established, this study demonstrates that there is a mutual interdependence between the rRNA modification process and large ribosome subunit assembly.

Plasticity and conditional essentiality of modification enzymes for domain V of Escherichia coli 23S ribosomal RNA.

Escherichia coli rRNAs are post-transcriptionally modified at 36 positions but their modification enzymes are dispensable individually for growth, bringing into question their significance. However, a major growth defect was reported for deletion of the RlmE enzyme, which abolished a 2'O methylation near the peptidyl transferase center (PTC) of the 23S rRNA. Additionally, an adjacent 80-nt "critical region" around the PTC had to be modified to yield significant peptidyl transferase activity in vitro. Surprisingly, we discovered that an absence of just two rRNA modification enzymes is conditionally lethal (at 20°C): RlmE and RluC. At a permissive temperature (37°C), this double knockout was shown to abolish four modifications and be defective in ribosome assembly, though not more so than the RlmE single knockout. However, the double knockout exhibited an even lower rate of tripeptide synthesis than did the single knockout, suggesting an even more defective ribosomal translocation. A combination knockout of the five critical-region-modifying enzymes RluC, RlmKL, RlmN, RlmM, and RluE (not RlmE), which synthesize five of the seven critical-region modifications and 14 rRNA and tRNA modifications altogether, was viable (minor growth defect at 37°C, major at 20°C). This was surprising based on prior in vitro studies. This five-knockout combination had minimal effects on ribosome assembly and frameshifting at 37°C, but greater effects on ribosome assembly and in vitro peptidyl transferase activity at cooler temperatures. These results establish the conditional essentiality of bacterial rRNA modification enzymes and also reveal unexpected plasticity of modification of the PTC region in vivo.

Ribosome Protein Composition Mediates Translation during the Escherichia coli Stationary Phase.

Bacterial ribosomes contain over 50 ribosome core proteins (r-proteins). Tens of non-ribosomal proteins bind to ribosomes to promote various steps of translation or suppress protein synthesis during ribosome hibernation. This study sets out to determine how translation activity is regulated during the prolonged stationary phase. Here, we report the protein composition of ribosomes during the stationary phase. According to quantitative mass-spectrometry analysis, ribosome core proteins bL31B and bL36B are present during the late log and first days of the stationary phase and are replaced by corresponding A paralogs later in the prolonged stationary phase. Ribosome hibernation factors Rmf, Hpf, RaiA, and Sra are bound to the ribosomes during the onset and a few first days of the stationary phase when translation is strongly suppressed. In the prolonged stationary phase, a decrease in ribosome concentration is accompanied by an increase in translation and association of translation factors with simultaneous dissociation of ribosome hibernating factors. The dynamics of ribosome-associated proteins partially explain the changes in translation activity during the stationary phase.

A mutation in the ribosomal protein uS12 reveals novel functions of its universally conserved PNSA loop.

The ribosomal protein uS12 is conserved across all domains of life. Recently, a heterozygous spontaneous mutation in human uS12 (corresponding to R49K mutation immediately downstream of the universally conserved 44 PNSA47 loop in Escherichia coli uS12) was identified for causing ribosomopathy, highlighting the importance of the PNSA loop. To investigate the effects of a similar mutation in the absence of any wild-type alleles, we mutated the rpsL gene (encoding uS12) in E. coli. Consistent with its pathology (in humans), we were unable to generate the R49K mutation in E. coli in the absence of a support plasmid. However, we were able to generate the L48K mutation in its immediate vicinity. The L48K mutation resulted in a cold sensitive phenotype and ribosome biogenesis defect in the strain. We show that the L48K mutation impacts the steps of initiation and elongation. Furthermore, the genetic interactions of the L48K mutation with RRF and Pth suggest a novel role of the PNSA loop in ribosome recycling. Our studies reveal new functions of the PNSA loop in uS12, which has so far been studied in the context of translation elongation.

Random pseuoduridylation in vivo reveals critical region of Escherichia coli 23S rRNA for ribosome assembly.

Pseudouridine is the most common modified nucleoside in RNA, which is found in stable RNA species and in eukaryotic mRNAs. Functional analysis of pseudouridine is complicated by marginal effect of its absence. We demonstrate that excessive pseudouridines in rRNA inhibit ribosome assembly. Ten-fold increase of pseudouridines in the 16S and 23S rRNA made by a chimeric pseudouridine synthase leads to accumulation of the incompletely assembled large ribosome subunits. Hyper modified 23S rRNA is found in the r-protein assembly defective particles and are selected against in the 70S and polysome fractions showing modification interference. Eighteen positions of 23S rRNA were identified where isomerization of uridines interferes with ribosome assembly. Most of the interference sites are located in the conserved core of the large subunit, in the domain 0 of 23S rRNA, around the peptide exit tunnel. A plausible reason for pseudouridine-dependent inhibition of ribosome assembly is stabilization of rRNA structure, which leads to the folding traps of rRNA and to the retardation of the ribosome assembly.

Toxins MazF and MqsR cleave Escherichia coli rRNA precursors at multiple sites.

The endoribonuclease toxins of the E. coli toxin-antitoxin systems arrest bacterial growth and protein synthesis by targeting cellular mRNAs. As an exception, E. coli MazF was reported to cleave also 16S rRNA at a single site and separate an anti-Shine-Dalgarno sequence-containing RNA fragment from the ribosome. We noticed extensive rRNA fragmentation in response to induction of the toxins MazF and MqsR, which suggested that these toxins can cleave rRNA at multiple sites. We adapted differential RNA-sequencing to map the toxin-cleaved 5'- and 3'-ends. Our results show that the MazF and MqsR cleavage sites are located within structured rRNA regions and, therefore, are not accessible in assembled ribosomes. Most of the rRNA fragments are located in the aberrant ribosomal subunits that accumulate in response to toxin induction and contain unprocessed rRNA precursors. We did not detect MazF- or MqsR-cleaved rRNA in stationary phase bacteria and in assembled ribosomes. Thus, we conclude that MazF and MqsR cleave rRNA precursors before the ribosomes are assembled and potentially facilitate the decay of surplus rRNA transcripts during stress.

SILAC analysis of Escherichia coli proteome during progression of growth from exponential to prolonged stationary phase.

The expression level of individual proteins varies markedly during the progression of the growth phase in bacteria. A set of proteins was quantified in Escherichia coli total proteome during 14 days of batch cultivation using pulse stable isotope labeled amino acids in cell culture (SILAC)-based quantitative mass spectrometry.

Ribosome degradation in growing bacteria.

Ribosomes are large ribozymes that synthesize all cellular proteins. As protein synthesis is rate-limiting for bacterial growth and ribosomes can comprise a large portion of the cellular mass, elucidation of ribosomal turnover is important to the understanding of cellular physiology. Although ribosomes are widely believed to be stable in growing cells, this has never been rigorously tested, owing to the lack of a suitable experimental system in commonly used bacterial model organisms. Here, we develop an experimental system to directly measure ribosomal stability in Escherichia coli. We show that (i) ribosomes are stable when cells are grown at a constant rate in the exponential phase; (ii) more than half of the ribosomes made during exponential growth are degraded during slowing of culture growth preceding the entry into stationary phase; and (iii) ribosomes are stable for many hours in the stationary phase. Ribosome degradation occurs in growing cultures that contain almost no dead cells and coincides with a reduction of comparable magnitude in the cellular RNA concentration.

Specificity and kinetics of 23S rRNA modification enzymes RlmH and RluD.

Along the ribosome assembly pathway, various ribosomal RNA processing and modification reactions take place. Stem-loop 69 in the large subunit of Escherichia coli ribosomes plays a substantial role in ribosome functioning. It contains three highly conserved pseudouridines synthesized by pseudouridine synthase RluD. One of the pseudouridines is further methylated by RlmH. In this paper we show that RlmH has unique substrate specificity among rRNA modification enzymes. It preferentially methylates pseudouridine and less efficiently uridine. Furthermore, RlmH is the only known modification enzyme that is specific to 70S ribosomes. Kinetic parameters determined for RlmH are the following: The apparent K(M) for substrate 70S ribosomes is 0.51 ± 0.06 µM, and for cofactor S-adenosyl-L-methionine 27 ± 3 µM; the k(cat) values are 4.95 ± 1.10 min⁻¹ and 6.4 ± 1.3 min⁻¹, respectively. Knowledge of the substrate specificity and the kinetic parameters of RlmH made it possible to determine the kinetic parameters for RluD as well. The K(M) value for substrate 50S subunits is 0.98 ± 0.18 µM and the k(cat) value is 1.97 ± 0.46 min⁻¹. RluD is the first rRNA pseudouridine synthase to be kinetically characterized. The determined rates of RluD- and RlmH-directed modifications of 23S rRNA are compatible with the rate of 50S assembly in vivo. The fact that RlmH requires 30S subunits demonstrates the dependence of 50S subunit maturation on the simultaneous presence of 30S subunits.

A Conundrum of r-Protein Stability: Unbalanced Stoichiometry of r-Proteins during Stationary Phase in Escherichia coli.

Bacterial ribosomes are composed of three rRNA and over 50 ribosomal protein (r-protein) molecules. r-proteins are essential for ribosome assembly and structural stability and also participate in almost all ribosome functions. Ribosomal components are present in stoichiometric amounts in the mature 70S ribosomes during exponential and early stationary growth phases. Ribosomes are degraded in stationary phase; however, the stability and fate of r-proteins during stationary growth phase are not known. In this study, we report a quantitative analysis of ribosomal components during extended stationary-phase growth in Escherichia coli. We show that (i) the quantity of ribosomes per cell mass decreases in stationary phase, (ii) 70S ribosomes contain r-proteins in stoichiometric amounts, (iii) 30S subunits are degraded faster than 50S subunits, (iv) the quantities of 21 r-proteins in the total proteome decrease during 14 days (short-lived r-proteins) concomitantly with the reduction of cellular RNA, and (e) 30 r-proteins are stable and form a pool of free r-proteins (stable r-proteins). Thus, r-proteins are present in nonstoichiometric amounts in the proteome of E. coli during the extended stationary phase. IMPORTANCE Ribosome degradation has been extensively described from the viewpoint of its main component, rRNA. Here, we aim to complement our knowledge by quantitatively analyzing r-protein degradation and stability both in the ribosomes and in the whole-cell proteome during stationary phase in E. coli. r-proteins are considered to be very stable in the proteome. Here, we show that a specific set of r-proteins are rapidly degraded after release from the rRNA. The degradation of r-proteins is an intriguing new aspect of r-protein metabolism in bacteria.

Ribosome reactivation by replacement of damaged proteins.

Ribosomal functions are vital for all organisms. Bacterial ribosomes are stable 2.4 MDa particles composed of three RNAs and over 50 different proteins. Accumulating damage to ribosomal RNA or proteins can disturb ribosome functioning. Organisms could benefit from degrading or possibly repairing inactive or partially active ribosomes. Reactivation of chemically damaged ribosomes by a process of protein replacement was studied in vitro. Ribosomes were inactivated by chemical modification of Cys residues. Incubation of modified ribosomes with total ribosomal proteins led to reactivation of translational activity. Intriguingly, ribosomal proteins extracted by LiCl are equally active in the restoration of ribosome function. Incubation of 70S ribosomes with isotopically labelled r-proteins followed by separation of ribosomes was used to identify exchangeable proteins. A similar set of proteins was found to be exchanged in vivo under stress conditions in the stationary phase. We propose that repair of damaged ribosomes might be an important mechanism for maintaining protein synthesis activity following chemical damage.

Identification of pseudouridine methyltransferase in Escherichia coli.

In ribosomal RNA, modified nucleosides are found in functionally important regions, but their function is obscure. Stem-loop 69 of Escherichia coli 23S rRNA contains three modified nucleosides: pseudouridines at positions 1911 and 1917, and N3 methyl-pseudouridine (m(3)Psi) at position 1915. The gene for pseudouridine methyltransferase was previously not known. We identified E. coli protein YbeA as the methyltransferase methylating Psi1915 in 23S rRNA. The E. coli ybeA gene deletion strain lacks the N3 methylation at position 1915 of 23S rRNA as revealed by primer extension and nucleoside analysis by HPLC. Methylation at position 1915 is restored in the ybeA deletion strain when recombinant YbeA protein is expressed from a plasmid. In addition, we show that purified YbeA protein is able to methylate pseudouridine in vitro using 70S ribosomes but not 50S subunits from the ybeA deletion strain as substrate. Pseudouridine is the preferred substrate as revealed by the inability of YbeA to methylate uridine at position 1915. This shows that YbeA is acting at the final stage during ribosome assembly, probably during translation initiation. Hereby, we propose to rename the YbeA protein to RlmH according to uniform nomenclature of RNA methyltransferases. RlmH belongs to the SPOUT superfamily of methyltransferases. RlmH was found to be well conserved in bacteria, and the gene is present in plant and in several archaeal genomes. RlmH is the first pseudouridine specific methyltransferase identified so far and is likely to be the only one existing in bacteria, as m(3)Psi1915 is the only methylated pseudouridine in bacteria described to date.

Phenotypic effects of paralogous ribosomal proteins bL31A and bL31B in E. coli.

Ribosomes are essential macromolecular complexes conducting protein biosynthesis in all domains of life. Cells can have heterogeneous ribosomes, i.e. ribosomes with various ribosomal RNA and ribosomal protein (r-protein) composition. However, the functional importance of heterogeneous ribosomes has remained elusive. One of the possible sources for ribosome heterogeneity is provided by paralogous r-proteins. In E. coli, ribosomal protein bL31 has two paralogs: bL31A encoded by rpmE and bL31B encoded by ykgM. This study investigates phenotypic effects of these ribosomal protein paralogs using bacterial strains expressing only bL31A or bL31B. We show that bL31A confers higher fitness to E. coli under lower temperatures. In addition, bL31A and bL31B have different effects on translation reading frame maintenance and apparent translation processivity in vivo as demonstrated by dual luciferase assay. In general, this study demonstrates that ribosomal protein paralog composition (bL31A versus bL31B) can affect cell growth and translation outcome.

Bacterial ribosome heterogeneity: Changes in ribosomal protein composition during transition into stationary growth phase.

Ribosomes consist of many small proteins and few large RNA molecules. Both components are necessary for ribosome functioning during translation. According to widely accepted view, bacterial ribosomes contain always the same complement of ribosomal proteins. Comparative bacterial genomics data indicates that several ribosomal proteins are encoded by multiple paralogous genes suggesting structural heterogeneity of ribosomes. In E. coli, two r-proteins bL31 and bL36 are encoded by two genes: rpmE and ykgM encode bL31 protein paralogs bL31A and bL31B, and rpmJ and ykgO encode bL36 protein paralogs bL36A and bL36B respectively. We have found several similarities and differences between ribosomes of exponential and stationary growth phases by using quantitative mass spectrometry and X-ray crystallography. First, composition of ribosome associating proteins changes profoundly as cells transition from exponential to stationary growth phase. Ribosomal core proteins bL31A and bL36A are replaced by bL31B and bL36B, respectively. Second, our X-ray structure of the 70S ribosome demonstrates that bL31B and bL36B proteins have similar ribosome binding sites to their A counterparts. Third, ribosome subpopulations containing A or B paralogs existed simultaneously demonstrating that E. coli ribosomes are heterogeneous with respect to their paralogous ribosomal protein composition that changes via protein exchange.

Substrate specificity of the pseudouridine synthase RluD in Escherichia coli.

Pseudouridine synthase RluD converts uridines at positions 1911, 1915, and 1917 of 23S rRNA to pseudouridines. These nucleotides are located in the functionally important helix-loop 69 of 23S rRNA. RluD is the only pseudouridine synthase that is required for normal growth in Escherichia coli. We have analyzed substrate specificity of RluD in vivo. Mutational analyses have revealed: (a) RluD isomerizes uridine in vivo only at positions 1911, 1915, and 1917, regardless of the presence of uridine at other positions in the loop of helix 69 of 23S rRNA variants; (b) substitution of one U by C has no effect on the conversion of others (i.e. formation of pseudouridines at positions 1911, 1915, and 1917 are independent of each other); (c) A1916 is the only position in the loop of helix 69, where mutations affect the RluD specific pseudouridine formation. Pseudouridines were determined in the ribosomal particles from a ribosomal large subunit defective strain (RNA helicase DeaD(-)). An absence of pseudouridines in the assembly precursor particles suggests that RluD directed isomerization of uridines occurs as a late step during the assembly of the large ribosomal subunit.

The Intersubunit Bridge B1b of the Bacterial Ribosome Facilitates Initiation of Protein Synthesis and Maintenance of Translational Fidelity.

In bacteria, ribosomal subunits are connected via 12 intersubunit bridges involving RNA-RNA, RNA-protein, and protein-protein interactions. The only protein-protein bridge in the ribosome is ribosomal intersubunit bridge 1b (B1b), which is mainly formed by the bacterial protein L31 (bL31) and connects the head domain of 30S subunit and the central protuberance of the 50S subunit. It is known to be the most dynamic intersubunit bridge. Here, we have evaluated the role of bL31 and thereby the bridge B1b in the working cycle of the ribosome. First, bL31-deficient ribosomes are severely compromised in their ability to ensure translational fidelity particularly in reading frame maintenance in vivo. Second, in the absence of bL31, the rate of initiation is significantly reduced both in vivo and in vitro. Third, polysome profile and subunit reassociation assays demonstrate that bL31 is important for stabilizing subunit joining in vivo and in vitro. Together, our results demonstrate that bL31 is important for determining translational fidelity and stabilizing subunit association. We conclude that the only protein-protein intersubunit bridge of the bacterial ribosome facilitates translation initiation and is essential for maintaining the reading frame of mRNA translation.

Analysis of the function of E. coli 23S rRNA helix-loop 69 by mutagenesis.

BACKGROUND: The ribosome is a two-subunit enzyme known to exhibit structural dynamism during protein synthesis. The intersubunit bridges have been proposed to play important roles in decoding, translocation, and the peptidyl transferase reaction; yet the physical nature of their contributions is ill understood. An intriguing intersubunit bridge, B2a, which contains 23S rRNA helix 69 as a major component, has been implicated by proximity in a number of catalytically important regions. In addition to contacting the small ribosomal subunit, helix 69 contacts both the A and P site tRNAs and several translation factors. RESULTS: We scanned the loop of helix 69 by mutagenesis and analyzed the mutant ribosomes using a plasmid-borne IPTG-inducible expression system. We assayed the effects of 23S rRNA mutations on cell growth, contribution of mutant ribosomes to cellular polysome pools and the ability of mutant ribosomes to function in cell-free translation. Mutations A1912G, and A1919G have very strong growth phenotypes, are inactive during in vitro protein synthesis, and under-represented in the polysomes. Mutation Psi1917C has a very strong growth phenotype and leads to a general depletion of the cellular polysome pool. Mutation A1916G, having a modest growth phenotype, is apparently defective in the assembly of the 70S ribosome. CONCLUSION: Mutations A1912G, A1919G, and Psi1917C of 23S rRNA strongly inhibit translation. Mutation A1916G causes a defect in the 50S subunit or 70S formation. Mutations Psi1911C, A1913G, C1914A, Psi1915C, and A1918G lack clear phenotypes.

Importance of transient structures during post-transcriptional refolding of the pre-23S rRNA and ribosomal large subunit assembly.

An important step of ribosome assembly is the folding of the rRNA into a functional structure. Despite knowledge of the folded state of rRNA in the ribosomal subunits, there is very little information on the rRNA folding pathway. We are interested in understanding how the functional structure of rRNA is formed and whether the rRNA folding intermediates have a role in ribosome assembly. To this end, transient secondary structures around both ends of pre-23S rRNA were analyzed by a chemical probing approach, using pre-23S rRNA transcripts. Metastable hairpin loop structures were found at both ends of 23S rRNA. The functional importance of the transient structures around the ends of 23S rRNA was tested by mutations that alter only the transient structure. The effect of mutations on 23S rRNA folding was tested in vitro and in vivo. It was found that both stabilization and destabilization of the transient structure around the 5' end of 23S rRNA inhibits post-transcriptional refolding in vitro and ribosome formation in vivo. The data suggest that the transient structure of rRNA has a function during 23S rRNA folding and thereby in ribosome assembly.

Functional interaction between RNase III and the Escherichia coli ribosome.

BACKGROUND: RNase III is a dsRNA specific endoribonuclease which is involved in the primary processing of rRNA and several mRNA species in bacteria. Both primary structural elements and the secondary structure of the substrate RNA play a role in cleavage specificity. RESULTS: We have analyzed RNase III cleavage sites around both ends of pre-23 S rRNA in the ribosome and in the protein-free pre-rRNA. It was found that in the protein-free pre-23 S rRNA the main cleavage site is at position (-7) in respect of the mature 5' end. When pre-23 S rRNA was in 70 S ribosomes or in 50 S subunits, the RNase III cleavage occurred at position (-3). We have demonstrated that RNase III interacts with both ribosomal subunits and with even higher affinity with 70 S ribosomes. Association of RNase III with 70 S ribosomes cannot be dissociated by poly(U) RNA indicating that the binding is specific. CONCLUSIONS: In addition to the primary and secondary structural elements in RNA, protein binding to substrate RNA can be a determinant of the RNase III cleavage site.

Different sensitivity of H69 modification enzymes RluD and RlmH to mutations in Escherichia coli 23S rRNA.

Nucleoside modifications are introduced into the ribosomal RNA during the assembly of the ribosome. The number and the localization of the modified nucleosides in rRNAs are known for several organisms. In bacteria, rRNA modified nucleosides are synthesized by a set of specific enzymes, the majority of which have been identified in Escherichia coli. Each rRNA modification enzyme recognizes its substrate nucleoside(s) at a specific stage of ribosome assembly. Not much is known about the specificity determinants involved in the substrate recognition of the modification enzymes. In order to shed light on the substrate specificity of RluD and RlmH, the enzymes responsible for the introduction of modifications into the stem-loop 69 (H69), we monitored the formation of H69 pseudouridines (Ψ) and methylated pseudouridine (m3Ψ) in vitro on ribosomes with alterations in 23S rRNA. While the synthesis of Ψs in H69 by RluD is relatively insensitive to the point mutations at neighboring positions, methylation of one of the Ψs by RlmH exhibited a much stronger sensitivity. Apparently, in spite of synthesizing modifications in the same region or even at the same position of rRNA, the two enzymes employ different substrate recognition mechanisms.