30S Ribosomal Assembly Research Articles

Parallel Pathways in 30S Ribosome Assembly

The bacterial 30S ribosomal subunit self-assembles in vitro to form an 850kDa RNP. Assembly is generally thought to advance when the core 16S rRNA navigates through many low-energy kinetic traps, guided by the 20 small-subunit proteins that recognize and lock in native RNA tertiary structure. Kinetic analyses of protein binding reveal a highly choreographed, ordered assembly process, consistent with large-scale qualitative observations of ordered protein binding and measurements of thermodynamic cooperativity within synthetic fragments of the 30S.

Concurrent nucleation of 16S folding and induced fit in 30S ribosome assembly

Rapidly growing cells produce thousands of new ribosomes each minute, in a tightly regulated process that is essential to cell growth. How the Escherichia coli 16S ribosomal RNA and the 20 proteins that make up the 30S ribosomal subunit can assemble correctly in a few minutes remains a challenging problem, partly because of the lack of real-time data on the earliest stages of assembly. By providing snapshots of individual RNA and protein interactions as they emerge in real time, here we show that 30S assembly nucleates concurrently from different points along the rRNA. Time-resolved hydroxyl radical footprinting was used to map changes in the structure of the rRNA within 20 milliseconds after the addition of total 30S proteins. Helical junctions in each domain fold within 100 ms. In contrast, interactions surrounding the decoding site and between the 5', the central and the 3' domains require 2-200 seconds to form. Unexpectedly, nucleotides contacted by the same protein are protected at different rates, indicating that initial RNA-protein encounter complexes refold during assembly. Although early steps in assembly are linked to intrinsically stable rRNA structure, later steps correspond to regions of induced fit between the proteins and the 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.

In vivo functional characterization of the Saccharomyces cerevisiae 60S biogenesis GTPase Nog1

The Saccharomyces cerevisiae Nog1 GTPase is critical for assembly of the large ribosomal subunit. Mutations in conserved residues in the GTP-binding pocket cause defects in cell growth and 60S ribosome assembly but mutant proteins retain their ability to associate with the pre-60S. Association of Nog1 with the pre-60S is independent of guanine nucleotide added to cell extracts. Thus, it appears that nucleotide occupancy does not substantially affect Nog1 association with pre-60S particles. Somewhat surprisingly, neither of the conserved threonines in the G2 motif of the GTPase domain is essential for Nog1 function. Neither the steady-state rRNA levels nor the protein composition (as determined by isobaric labeling and identification by mass spectrometry of peptides) of the pre-60S particles in the nog1P176V mutant are grossly perturbed, although levels of four proteins (Nog1, Nop2, Nop15, and Tif6) are modestly reduced in pre-60S particles isolated from the mutant. Deletion analysis revealed that the C-terminal 168 amino acids are not required for function; however, the N-terminal 126 amino acids are required. Optimal association with pre-60S particles requires sequences between amino acids 347-456. Several conserved charge-to-alanine substitutions outside the GTPase domain display modest growth phenotypes indicating that these residues are not critical for function.

The p21-Activated Protein Kinase Inhibitor Skb15 and Its Budding Yeast Homologue Are 60S Ribosome Assembly Factors

Ribosome biogenesis is driven by a large number of preribosomal factors that associate with and dissociate from the preribosomal particles along the maturation pathway. We have previously shown that budding yeast Mak11, whose homologues in other eukaryotes were described as modulating a p21-activated protein kinase function, accumulates in Rlp24-associated pre-60S complexes when their maturation is impeded in Saccharomyces cerevisiae. The functional inactivation of WD40 repeat protein Mak11 interfered with the 60S rRNA maturation, led to a cell cycle delay in G(1), and blocked green fluorescent protein-tagged Rpl25 in the nucleoli of yeast cells, indicating an early role of Mak11 in ribosome assembly. Surprisingly, Mak11 inactivation also led to a dramatic destabilization of Rlp24. The suppression of the thermosensitive phenotype of a mak11 mutant by RLP24 overexpression and a direct in vitro interaction between Rlp24 and Mak11 suggest that Mak11 acts as an Rlp24 cofactor during early steps of 60S ribosomal subunit assembly. Moreover, we found that Skb15, the Mak11 homologue in Schizosaccharomyces pombe, also associated with preribosomes and affected 60S biogenesis in fission yeast. It is thus likely that the previously observed phenotypes for MAK11 homologues in other eukaryotes are secondary to the main function of these proteins in ribosome formation.

Mapping contacts of the S12–S7 intercistronic region of str operon mRNA with ribosomal protein S7 of E. coli

In E. coli, S7 initiates 30S ribosome assembly by binding to 16S rRNA. It also regulates translation of the S12 and S7 cistrons of the ‘streptomycin’ operon transcript by binding to the S12–S7 intercistronic region. Here, we describe the contacts of N-terminally His 6-tagged S7 with this region as mapped by UV-induced cross-linking. The cross-links are located at U(−34), U(−35), quite distant from the start codons of the two cistrons. In order to explain the mechanism of translational repression of S12–S7, we consider a possible conformational rearrangement of the intercistronic RNA structure induced by S7 binding.

Ribosome assembly reawakens

Ribosome assembly is a topic that causes many cell biology students to yawn, for it is often taught as a series of somewhat untantalizing steps plodding towards the formation of a complex mass of RNA and protein that carries out the important and well-defined job of translating mRNA into protein. Recently, however, ribosome biogenesis has jumped into the spotlight with the finding that a 60S ribosome assembly factor, Yph1/Nop7p, is found associated with the origin-recognition complex (ORC) and with other interesting proteins that participate in cell-cycle regulation [ 1. Adams C.C. et al. Saccharomyces cerevisiae nucleolar protein Nop7p is necessary for biogenesis of 60S ribosomal subunits. RNA. 2002; 8: 150-165 Crossref PubMed Scopus (74) Google Scholar ].

Lack of complete cooperativity of ribosome assembly in vitro and its possible relevance to in vivo ribosome assembly and the regulation of ribosomal gene expression

Earlier studies have shown that the reconstitution of Escherichia coli 50S as well as 30S ribosomal subunits from component rRNA and ribosomal protein (r-protein) molecules in vitro is not completely cooperative and binding of more than one r-protein to a single 16S rRNA (or 23S rRNA) molecule is required to initiate a successful 30S (or 50S) ribosome assembly reaction. We first confirmed this conclusion by carrying out 30S subunit reconstitution in the presence of a constant amount of 16S rRNA together with various amounts of total 30S r-proteins (TP30) and by analyzing the physical state of reconstituted particles rather than by assaying protein synthesizing activity of the particles as was done in the earlier studies. As expected, under conditions of excess rRNA, the efficiency of 30S subunit reconstitution per unit amount of TP30 decreased greatly with the decrease in the ration of TP30 to rRNA, indicating the lack of complete cooperativity in the assembly reaction. We then asked the question whether the cooperativity of ribosome assembly is complete in vivo. We treated exponentially growing E coli cells with low concentrations of chloramphenicol which is known to inhibit protein synthesis without inhibiting rRNA synthesis, creating conditions of excess synthesis of rRNA relative to r-proteins. Several concentrations of chloramphenicol (ranging from 0.4 to 4.0 μg/ml) were used so that inhibition of protein synthesis ranged from 40 to 95%. Under these conditions, we examined the synthesis of RNA, ribosomal proteins and 50S ribosomal subunits as well as the synthesis of total protein. We found that the synthesis of 50S subunits was not inhibited as much as the synthesis of total protein at lower concentrations of chloramphenicol, but the degree of inhibition of 50S subunit synthesis increased sharply with increasing concentrations of chloramphenicol and was in fact greater than the degree of inhibition of total protein synthesis at chloramphenicol concentrations of 2 μg/ml or higher. The inhibition of 50S subunit synthesis was significantly greater than the inhibition of r-protein synthesis at all chloramphenicol concentrations examined. These data are consistent with the hypothesis that the cooperativity of ribosome assembly in vivo is also not complete as in the case for in vitro ribosome reconstitution, but are difficult, if not impossible, to explain on the basis of the complete cooperativity model. It is suggested that the two major regulatory mechanisms of rRNA synthesis in E coli, stringent control and growth-rate-dependent control, may have evolved to prevent overproduction of rRNA relative to r-proteins, thus preventing possible breakdown of cooperative assembly of ribosomes under conditions of high rRNA/r-protein ratios.

Specific ribosomal RNA recognition by a fragment of E. coli ribosomal protein S4 missing the C-terminal 36 amino acid residues.

We have previously investigated the role of the N-terminal region of ribosomal protein S4 to participate in 30S ribosome assembly and function (1-3). In this report we extend these studies to the two fragments produced by the chemical cleavage of protein S4 at the tryptophan residue 167. We find that the carboxyl terminal fragment (168-203) does not bind 16S RNA nor does it participate in assembly with the other 20 proteins from the 30S ribosome. In contrast, the larger fragment (1-167), does bind 16S RNA specifically. If the S4-fragment (1-167) is used to replace protein S4 in the complete 30S assembly reaction, all 20 of the other 30S proteins are incorporated. We conclude that the carboxyl terminal section of the protein S4 is not directly involved in binding 16S RNA or in the assembly of any of the other 30S proteins.

Analysis of protein-protein relationships in 30S ribosome assembly intermediates using protection from proteolytic digestion

Treatment of the intact bacterial ribosome with proteolytic enzymes results in little or no digestion of many of the component proteins [Craven, G. R., & Gupta, V. (1970) Proc. Natl. Acad. Sci. U.S.A. 67, 1329]. In contrast, when the proteins are released from the constraints of ribosome structure they become completely susceptible to proteolytic attack. We have attempted to exploit these observations in an effort to determine the precise steps in ribosome assembly which result in a conversion of the structures of the various proteins from a proteolysis sensitive to a resistant form. Thus, a total of 11 30S ribosome assembly intermediate complexes of proteins and 16S RNA were prepared and digested with trypsin or chymotrypsin. The kinetics of digestion of each protein in the complex were followed by polyacrylamide gel electrophoresis. By a comparison of the digestion pattern of two complexes differing only by the presence of a single protein, it was possible to deduce several specific protective effects of one protein on its neighbor in the complex. On the basis of these studies, we propose nine protein-protein protective effects. The possible relevance of these interrelationships to other well-established proximity relationships is discussed.

Studies on the ability of partially iodinated 16S RNA to participate in 30S ribosome assembly.

Deproteinated 16S RNA was iodinated at pH 5.0 in an aqueous solution containing TlCl3 plus KI for 1-5 hours at 42 degrees C. Under these conditions 33 moles of iodine are incorporated per mole of RNA. As judged by sucrose gradient sedimentation, the iodinated RNA does not exhibit any large alteration in conformation as compared to unmodified 16S. The iodinated RNA was examined for its ability to reconstitute with total 30S proteins. Sedimentation velocity analysis reveals that the reconstituted subunit has a sedimentation constant of approximately 20S. In addition, protein analysis of particles reconstituted with 16S RNA iodinated for 5 hours indicates that proteins S2, S10, S13, S14, S15, S17, S18, S19, and S21 are no longer able to participate in the 30S assembly process and that proteins S6, S16 and S20 are present in reduced amounts. The ramifications of these results concerning protein-RNA and RNA-RNA interactions occurring in ribosome assembly are discussed.