Ribosomal protein-dependent orientation of the 16 S rRNA environment of S15

Ribosomal protein S15 binds specifically to the central domain of 16 S ribosomal RNA (16 S rRNA) and directs the assembly of four additional proteins to this domain. The central domain of 16 S rRNA along with these five proteins form the platform of the 30 S subunit. Previously, directed hydroxyl radical probing from Fe(II)-S15 in small ribonucleoprotein complexes was used to study assembly of the central domain of 16 S rRNA. Here, this same approach was used to understand the 16 S rRNA environment of Fe(II)-S15 in 30 S subunits and to determine the ribosomal proteins that are involved in forming the mature S15-16 S rRNA environment. We have identified additional sites of Fe(II)-S15-directed cleavage in 30S subunits compared to the binary complex of Fe(II)-S15/16 S rRNA. Along with novel targets in the central domain, sites within the 5' and 3' minor domains are also cleaved. This suggests that during the course of 30S subunit assembly these elements are positioned in the vicinity of S15. Besides the previously determined role for S8, roles for S5, S6+S18, and S16 in altering the 16 S rRNA environment of S15 were established. These studies reveal that ribosomal proteins can alter the assembly of regions of the 30 S subunit from a considerable distance and influence the overall conformation of this ribonucleoprotein particle.     

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.     

Assembly of the 5' and 3' minor domains of 16S ribosomal RNA as monitored by tethered probing from ribosomal protein S20

The ribosomal protein (r-protein) S20 is a primary binding protein. As such, it interacts directly and independently with the 5′ domain as well as the 3′ minor domain of 16S ribosomal RNA (rRNA) in minimal particles and the fully assembled 30S subunit. The interactions observed between r-protein S20 and the 5′ domain of 16S rRNA are quite extensive, while those between r-protein S20 and the 3′ minor domain are significantly more limited. In this study, directed hydroxyl radical probing mediated by Fe(II)-derivatized S20 proteins was used to monitor the folding of 16S rRNA during r-protein association and 30S subunit assembly. An analysis of the cleavage patterns in the minimal complexes [16S rRNA and Fe(II)-S20] and the fully assembled 30S subunit containing the same Fe(II)-derivatized proteins shows intriguing similarities and differences. These results suggest that the two domains, 5′ and 3′ minor, are organized relative to S20 at different stages of assembly. The 5′ domain acquires, in a less complex ribonucleoprotein particle than the 3′ minor domain, the same architecture as observed in mature subunits. These results are similar to what would be predicted of subunit assembly by the 5′-to-3′ direction assembly model.     

Structural insights into methyltransferase KsgA function in 30S ribosomal subunit biogenesis

The assembly of the ribosomal subunits is facilitated by ribosome biogenesis factors. The universally conserved methyltransferase KsgA modifies two adjacent adenosine residues in the 3'-terminal helix 45 of the 16 S ribosomal RNA (rRNA). KsgA recognizes its substrate adenosine residues only in the context of a near mature 30S subunit and is required for the efficient processing of the rRNA termini during ribosome biogenesis. Here, we present the cryo-EM structure of KsgA bound to a nonmethylated 30S ribosomal subunit. The structure reveals that KsgA binds to the 30S platform with the catalytic N-terminal domain interacting with substrate adenosine residues in helix 45 and the C-terminal domain making extensive contacts to helix 27 and helix 24. KsgA excludes the penultimate rRNA helix 44 from adopting its position in the mature 30S subunit, blocking the formation of the decoding site and subunit joining. We suggest that the activation of methyltransferase activity and subsequent dissociation of KsgA control conformational changes in helix 44 required for final rRNA processing and translation initiation.     

The role of RbfA in 16S rRNA processing and cell growth at low temperature in Escherichia coli

RbfA, a 30S ribosome-binding factor, is a multicopy suppressor of a cold-sensitive C23U mutation of the 16S rRNA and is required for efficient processing of the 16S rRNA. At 37 degrees C, DeltarbfA cells show accumulation of ribosomal subunits and 16S rRNA precursor with a significantly reduced polysome profile in comparison with wild-type cells. RbfA is also a cold-shock protein essential for Escherichia coli cells to adapt to low temperature. In this study, we examined its association with the ribosome and its role in 16S rRNA processing and ribosome profiles at low temperature. In wild-type cells, following cold shock at 15 degrees C, the amount of free RbfA remained largely stable, while that of its 30S subunit-associated form became several times greater than that at 37 degrees C and a larger fraction of total 30S subunits was detected to be RbfA-containing. In DeltarbfA cells, the pre-16S rRNA amount increased after cold shock with a concomitant reduction of the mature 16S rRNA amount and the formation of polysomes was further reduced. A closer examination revealed that 30S ribosomal subunits of DeltarbfA cells at low temperature contained primarily pre-16S rRNA and little mature 16S rRNA. Our results indicate that the cold sensitivity of DeltarbfA cells is directly related to their lack of translation initiation-capable 30S subunits containing mature 16S rRNA at low temperature. Importantly, when the C-terminal 25 residue sequence was deleted, the resulting RbfADelta25 lost the abilities to stably associate with the 30S subunit and to suppress the dominant-negative, cold-sensitive phenotype of the C23U mutation in 16S rRNA but was able to suppress the 16S rRNA processing defect and the cold-sensitive phenotype of the DeltarbfA cells, suggesting that RbfA may interact with the 30S ribosome at more than one site or function in more than one fashion in assisting the 16S rRNA maturation at low temperature.     

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.     

Nature of the ribosomal binding site for initiation factor 3 (IF-3)

$\text{In vitro}$ labelled IF-3 binds to both 16S and 23S rRNA but while one molecule of IF-3 binds to each 30S particle, binding to 50S particles is negligible. If proteins are removed by LiCl or CsCl treatment from either ribosomal subunit, however, binding specificity is lost and new “binding sites” appear on both ribosomal particles. Controlled RNase digestion of the 30S subunits does not cause the loss of any r-protein while controlled trypsin digestion results in the loss or degradation of several r-proteins; compared to the Phe-tRNA binding site, the binding site of IF-3 seems to be more sensitive to RNase than to trypsin digestion. Antibodies against single 30S r-proteins, which inhibit other ribosomal functions, do not prevent the binding of IF-3. RNA-binding dyes (acridine orange and pyronine) inhibit the binding of IF-3 to 30S ribosomal subunits. It is proposed that a segment of the 16S rRNA provides the binding site for IF-3 and that r-proteins confer specificity, restricting the number of available “binding sites”, and stabilize the 30S-IF-3 interaction.     

Stepwise dissociation of yeast 60S ribosomal subunits by LiCl and identification of L25 as a primary 26S rRNA binding protein

Treatment of yeast 60S ribosomal subunits with 0.5 M LiCl was found to remove all but six of the ribosomal proteins. The proteins remaining associated with the (26S + 5.8S) rRNA complex were identified as L4, L8, L10, L12, L16 and L25. These core proteins were split off sequentially in the order (L16 + L12), L10, (L4 + L8), L25 by further increasing the LiCl concentration. At 1.0 M LiCl only ribosomal protein L25 remains bound to the rRNA. Upon lowering the LiCl concentration the core proteins reassociate with the rRNA in the reverse order of their removal. The susceptibility of the ribosomal proteins to removal by LiCl corresponds quite well with their order of assembly into the 60S subunit in vivo as determined earlier [Kruiswijk et al. (1978) Biochim. Biophys. Acta 517, 378-389]. Binding studies in vitro using partially purified L25 showed that this protein binds specifically to 26S rRNA. Therefore our experiments for the first time directly identify a eukaryotic ribosomal protein capable of binding to high-molecular-mass rRNA. Binding studies in vitro using a blot technique demonstrated that core proteins L8 and L16 as well as protein L21, though not present in any of the core particles, are also capable of binding to 26S rRNA to approximately the same extent as L25. About nine additional 60S proteins appeared to interact with the 26S rRNA, though to a lesser extent.     

Nonbridging phosphate oxygens in 16S rRNA important for 30S subunit assembly and association with the 50S ribosomal subunit

Ribosomes are composed of RNA and protein molecules that associate together to form a supramolecular machine responsible for protein biosynthesis. Detailed information about the structure of the ribosome has come from the recent X-ray crystal structures of the ribosome and the ribosomal subunits. However, the molecular interactions between the rRNAs and the r-proteins that occur during the intermediate steps of ribosome assembly are poorly understood. Here we describe a modification-interference approach to identify nonbridging phosphate oxygens within 16S rRNA that are important for the in vitro assembly of the Escherichia coli 30S small ribosomal subunit and for its association with the 50S large ribosomal subunit. The 30S small subunit was reconstituted from phosphorothioate-substituted 16S rRNA and small subunit proteins. Active 30S subunits were selected by their ability to bind to the 50S large subunit and form 70S ribosomes. Analysis of the selected population shows that phosphate oxygens at specific positions in the 16S rRNA are important for either subunit assembly or for binding to the 50S subunit. The X-ray crystallographic structures of the 30S subunit suggest that some of these phosphate oxygens participate in r-protein binding, coordination of metal ions, or for the formation of intersubunit bridges in the mature 30S subunit. Interestingly, however, several of the phosphate oxygens identified in this study do not participate in any interaction in the mature 30S subunit, suggesting that they play a role in the early steps of the 30S subunit assembly.     

Understanding ribosome assembly: the structure of in vivo assembled immature 30S subunits revealed by cryo-electron microscopy

Four decades after early in vitro assembly studies demonstrated that ribosome assembly is a controlled process, our understanding of ribosome assembly is still incomplete. Just as structure determination has been so important to understanding ribosome function, so too will it be critical to sorting out the assembly process. Here, we used a viable deletion in the yjeQ gene, a recognized ribosome assembly factor, to isolate and structurally characterize immature 30S subunits assembled in vivo. These small ribosome subunits contained unprocessed 17S rRNA and lacked some late ribosomal proteins. Cryo-electron microscopy reconstructions revealed that the presence of precursor sequences in the rRNA induces a severe distortion in the 3' minor domain of the subunit involved in the decoding of mRNA and interaction with the large ribosome subunit. These findings suggest that rRNA processing events induce key local conformational changes directing the structure toward the mature assembly. We concluded that rRNA processing, folding, and the entry of tertiary r-proteins are interdependent events in the late stages of 30S subunit assembly. In addition, we demonstrate how studies of emerging assembly factors in ribosome biogenesis can help to elucidate the path of subunit assembly in vivo.     

The RimP Protein Is Important for Maturation of the 30S Ribosomal Subunit

The in vivo assembly of ribosomal subunits requires assistance by auxiliary proteins that are not part of mature ribosomes. More such assembly proteins have been identified for the assembly of the 50S than for the 30S ribosomal subunit. Here, we show that the RimP protein (formerly YhbC or P15a) is important for the maturation of the 30S subunit. A rimP deletion (ΔrimP135) mutant in Escherichia coli showed a temperature-sensitive growth phenotype as demonstrated by a 1.2-, 1.5-, and 2.5-fold lower growth rate at 30, 37, and 44 °C, respectively, compared to a wild-type strain. The mutant had a reduced amount of 70S ribosomes engaged in translation and showed a corresponding increase in the amount of free ribosomal subunits. In addition, the mutant showed a lower ratio of free 30S to 50S subunits as well as an accumulation of immature 16S rRNA compared to a wild-type strain, indicating a deficiency in the maturation of the 30S subunit. All of these effects were more pronounced at higher temperatures. RimP was found to be associated with free 30S subunits but not with free 50S subunits or with 70S ribosomes. The slow growth of the rimP deletion mutant was not suppressed by increased expression of any other known 30S maturation factor.     

Assembly of the central domain of the 30S ribosomal subunit: roles for the primary binding ribosomal proteins S15 and S8

Assembly of the 30S ribosomal subunit occurs in a highly ordered and sequential manner. The ordered addition of ribosomal proteins to the growing ribonucleoprotein particle is initiated by the association of primary binding proteins. These proteins bind specifically and independently to 16S ribosomal RNA (rRNA). Two primary binding proteins, S8 and S15, interact exclusively with the central domain of 16S rRNA. Binding of S15 to the central domain results in a conformational change in the RNA and is followed by the ordered assembly of the S6/S18 dimer, S11 and finally S21 to form the platform of the 30S subunit. In contrast, S8 is not part of this major platform assembly branch. Of the remaining central domain binding proteins, only S21 association is slightly dependent on S8. Thus, although S8 is a primary binding protein that extensively contacts the central domain, its role in assembly of this domain remains unclear. Here, we used directed hydroxyl radical probing from four unique positions on S15 to assess organization of the central domain of 16S rRNA as a consequence of S8 association. Hydroxyl radical probing of Fe(II)-S15/16S rRNA and Fe(II)-S15/S8/16S rRNA ribonucleoprotein particles reveal changes in the 16S rRNA environment of S15 upon addition of S8. These changes occur predominantly in helices 24 and 26 near previously identified S8 binding sites. These S8-dependent conformational changes are consistent with 16S rRNA folding in complete 30S subunits. Thus, while S8 binding is not absolutely required for assembly of the platform, it appears to affect significantly the 16S rRNA environment of S15 by influencing central domain organization.     

Precursor Ribosomal Ribonucleic Acid and Ribosome Accumulation In Vivo During the Recovery of Salmonella typhimurium from Thermal Injury

When cells of S. typhimurium were heated at 48 C for 30 min in phosphate buffer (pH 6.0), they became sensitive to Levine Eosin Methylene Blue Agar containing 2% NaCl (EMB-NaCl). The inoculation of injured cells into fresh growth medium supported the return of their normal tolerance to EMB-NaCl within 6 hr. The fractionation of ribosomal ribonucleic acid (rRNA) from unheated and heat-injured cells by polyacrylamide gel electrophoresis demonstrated that after injury the 16S RNA species was totally degraded and the 23S RNA was partially degraded. Sucrose gradient analysis demonstrated that after injury the 30S ribosomal subunit was totally destroyed and the sedimentation coefficient of the 50S particle was decreased to 47S. During the recovery of cells from thermal injury, four species of rRNA accumulated which were demonstrated to have the following sedimentation coefficients: 16, 17, 23, and 24S. Under identical recovery conditions, 22, 26, and 28S precursors of the 30S ribosomal subunit and 31 and 48S precursors of the 50S ribosomal subunit accumulated along with both the 30 and 50S mature particles. The addition of chloramphenicol to the recovery medium inhibited both the maturation of 17S RNA and the production of mature 30S ribosomal subunits, but permitted the accumulation of a single 22S precursor particle. Chloramphenicol did not affect either the maturation of 24S RNA or the mechanism of formation of 50S ribosomal subunits during recovery. Very little old ribosomal protein was associated with the new rRNA synthesized during recovery. New ribosomal proteins were synthesized during recovery and they were found associated with the new rRNA in ribosomal particles. The rate-limiting step in the recovery of S. typhimurium from thermal injury was in the maturation of the newly synthesized rRNA.     

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.     

Binding of 16S rRNA to chloroplast 30S ribosomal proteins blotted on nitrocellulose

Protein-RNA associations were studied by a method using proteins blotted on a nitrocellulose sheet. This method was assayed with Escherichia Coli 30S ribosomal components. In stringent conditions (300 mM NaCl or 20° C) only 9 E. coli ribosomal proteins strongly bound to the 16S rRNA: S4, S5, S7, S9, S12, S13, S14, S19, S20. 8 of these proteins have been previously found to bind independently to the 16S rRNA. The same method was applied to determine protein-RNA interactions in spinach chloroplast 30S ribosomal subunits. A set of only 7 proteins was bound to chloroplast rRNA in stringent conditions: chloroplast S6, S10, S11, S14, S15, S17 and S22. They also bound to E. coli 16S rRNA. This set includes 4 chloroplast-synthesized proteins: S6, S11, S15 and S22. The core particles obtained after treatment by LiCl of chloroplast 30S ribosomal subunit contained 3 proteins (S6, S10 and S14) which are included in the set of 7 binding proteins. This set of proteins probably play a part in the early steps of the assembly of the chloroplast 30S ribosomal subunit.     

Analysis of conformational changes in 16 S rRNA during the course of 30 S subunit assembly

Ribosome biogenesis involves an integrated series of binding events coupled with conformational changes that ultimately result in the formation of a functional macromolecular complex. In vitro, Escherichia coli 30 S subunit assembly occurs in a cooperative manner with the ordered addition of 20 ribosomal proteins (r-proteins) with 16 S rRNA. The assembly pathway for 30 S subunits has been dissected in vitro into three steps, where specific r-proteins associate with 16 S rRNA early in 30 S subunit assembly, followed by a mid-assembly conformational rearrangement of the complex that then enables the remaining r-proteins to associate in the final step. Although the three steps of 30 S subunit assembly have been known for some time, few details have been elucidated about changes that occur as a result of these three specific stages. Here, we present a detailed analysis of the concerted early and late stages of small ribosomal subunit assembly. Conformational changes, roles for base-pairing and r-proteins at specific stages of assembly, and a polar nature to the assembly process have been revealed. This work has allowed a more comprehensive and global view of E.coli 30 S ribosomal subunit assembly to be obtained.     

Halobacterium cutirubrum ribosomes. Properties of the ribosomal proteins and ribonucleic acid

1. The 30S ribosomal subunit of the extreme halophile Halobacterium cutirubrum is unstable and loses 75% of its ribosomal protein when the 70S ribosome is dissociated into the two subunits. A stable 30S subunit is obtained if the dissociation of the 70S particle is carried out in the presence of the soluble fraction. 2. A fractionation procedure was developed for the selective removal of groups of proteins from the 30S and 50S subunits. When the ribosomes, which are stable in 4m-K(+) and 0.1m-Mg(2+), were extracted with low-ionic-strength buffer 75-80% of the 30S proteins and 60-65% of the 50S proteins as well as the 5S rRNA were released. The proteins in this fraction are the most acidic of the H. cutirubrum ribosomal proteins. Further extraction with Li(+)-EDTA releases additional protein, leaving a core particle containing either 16S rRNA or 23S rRNA and about 5% of the total ribosomal protein. The amino acid composition, mobility on polyacrylamide gels at pH4.5 and 8.7, and the molecular-weight distribution of the various protein fractions were determined. 3. The s values of the rRNA are 5S, 16S and 23S. The C+G contents of the 16S and 23S rRNA were 56.1 and 58.8% respectively and these are higher than C+G contents of the corresponding Escherichia coli rRNA (53.8 and 54.1%).     

On the interpretation of small-angle x-ray solution scattering from spherical viruses.

The intermediates in the ribosome assembly in exponentially growing Escherichia coli have been identified by centrifuging a crude lysate, pulse-labeled with a radioactive RNA base, through a sucrose gradient and analyzing for precursor rRNA in the gradient fractions by gel electrophoresis. The major intermediate in the assembly of the 50 S subunit cosediments with the mature subunit, whereas two minor precursor species sediment between the 30 S and 50 S peaks. The assembly of the 30 S subunit proceeds via a minor intermediate sedimenting slightly behind the mature subunit and a major precursor particle that cosediments with the mature 30 S subunit.The fraction of the rRNA contained in these precursor particles was determined by direct determination of the amount of rRNA in the precursor particles, and from the labeling kinetics of their rRNA. The direct estimation indicated that about 2% of the total 23 S type RNA, and 3 to 5% of the total 16 S type RNA is harboured in precursor particles. In the kinetic experiments the specific activity of the nucleoside triphosphates and of the different ribosomal particles was followed after addition of a radioactive RNA precursor to the growth medium. The results were compared with a digital simulation of the flow of isotopes through the assembly pathways. This method indicated that approximately 2% of the total 23 S type RNA, as well as 2% of the total 16 S type RNA, is contained in the precursor particles.