The extended loops of ribosomal proteins L4 and L22 are not required for ribosome assembly or L4-mediated autogenous control

Ribosomal proteins L4 and L22 both have a globular domain that sits on the surface of the large ribosomal subunit and an extended loop that penetrates its core. The tips of both loops contribute to the lining of the peptide exit tunnel and have been implicated in a gating mechanism that might regulate the exit of nascent peptides. Also, the extensions of L4 and L22 contact multiple domains of 23S rRNA, suggesting they might facilitate rRNA folding during ribosome assembly. To learn more about the roles of these extensions, we constructed derivatives of both proteins that lack most of their extended loops. Our analysis of ribosomes carrying L4 or L22 deletion proteins did not detect any significant difference in their sedimentation property or polysome distribution. Also, the role of L4 in autogenous control was not affected. We conclude that these extensions are not required for ribosome assembly or for L4-mediated autogenous control of the S10 operon.     

On the sequence homology of the ribosomal proteins, Escherichia coli S11, yeast rp59 and Chinese hamster S14

A strong sequence homology was found among the ribosomal proteins of the different species, S11 of Escherichia coli, rp59 of Saccharomyces cerevisiae and S14 of Chinese hamster ovary cell. The significance of this series of highly conserved proteins is discussed.     

Methylation of ribosomal proteins during ribosome assembly in Escherichia coli

Summary In Escherichia coli, a number of ribosomal proteins are methylated. The time of methylation of L7 and L11 during ribosome assembly was studied. It was observed that the methylation of L7 could occur in the free protein stage. Both the 32S and 40S ribonucleoprotein intermediates also contained methylated L7 although the extent of methylation in these particles was not as high as in the free L7, the 45S or the 50S particles. Free L11 could also be partially methylated but the bulk of methylation of this protein was found in the 45S and the 50S particles.It was previously reported that the methylation of L7 is inversely proportional to the growth temperature (Chang 1978), we now show that once L7 is methylated at 25°, the methyl group is stable when the culture is shifted to 37°C. However, a partial turnover of the methyl group of L7 is observed when the methylated ribosome is chased at 25°C. On the other hand, the methyl groups of L11 appear to be stable at either 25°C or 37°C. We also observe that the extent of methylation of both L7 and L11 stays nearly constant during the cell growth cycle from early log to stationary phase.     

Inhibition of synthesis of ribosomal proteins and of ribosome assembly after infection of L cells with vesicular stomatitis virus

The effect of infection of mouse L cells by vesicular stomatitis virus on the synthesis of ribosomal proteins was investigated using two-dimensional polyacrylamide gel electrophoresis to analyze the ribosomal proteins. It was found that the synthesis of nearly all of the cytoplasmic ribosomal proteins examined was inhibited by infection and mostly to the same extent. Analysis of the ribosomal proteins extracted from intact ribosomes indicated that infection also reduces the incorporation of all the ribosomal proteins tested into assembled ribosomes. The inhibition of ribosome assembly was greater than the inhibition of synthesis of ribosomal proteins, suggesting that some other factor was also limiting the assembly of ribosomes. As shown in this report, infection also inhibits ribosomal RNA production. Thus, the decreased assembly of ribosomes in infected cells probably results from the inhibition of synthesis of both ribosomal proteins and ribosomal RNA.     

Mitochondrial ribosome assembly in Neurospora. Two-dimensional gel electrophoretic analysis of mitochondrial ribosomal proteins

Recent results with Neurospora crassa show that one protein (S-5, mol wt 52,000) associated with the mitochondrial (mit) small ribosomal subunit is translated within the mitochondria (Lambowitz et al. 1976. J. Mol. Biol. 107:223-253). In the present work, Neurospora mit ribosomal proteins were analyzed by two-dimensional gel electrophoresis using a modification of the gel system of Mets and Bogorad. The results show that S-5 is present in near stoichiometric concentrations in high salt (0.5 MKCl)-washed mit small subunits from wild-type strains. S-5 is among the most basic mit ribosomal proteins (pI greater than 10) and has a high affinity for RNA under the conditions of the urea-containing gel buffers. The role of S-5 in mit ribosome assembly was investigated by an indirect method, making use of chloramphenicol to specifically inhibit mit protein synthesis. Chloramphenicol was found to rapidly inhibit the assembly of mit small subunits leading to the formation of CAP-30S particles which sediment slightly behind mature small subunits (LaPolla and Lambowitz. 1977. J. Mol. 116: 189-205). Two-dimensional gel analysis shows that the more slowly sedimentaing CAP-30S particles are deficient in S-5 and in several other proteins, whereas these proteins are present in normal concentrations in mature small subunits from the same cells. Because S-5 is the only mit ribosomal protein whose synthesis is directly inhibited by chloramphenicol, the results tentatively suggest that S-5 plays a role in the assembly of mit small subunits. In addition, the results are consistent with the idea that S-5 stabilizes the binding of several other mit small subunit proteins. Two-dimensional gel electrophoresis was used to examine mit ribosomal proteins from [poky] and six additional extra-nuclear mutants with defects in the assembly of mit small subunits. The electrophoretic mobility of S-5 is not detectably altered in any of the mutants. However, [poky] mit small subunits are deficient in S-5 and also contain several other proteins in abnormally low or high concentrations. These and other results are consistent with a defect in a mit ribosomal constituent in [poky].     

Functional conservation between structurally diverse ribosomal proteins from Drosophila melanogaster and Saccharomyces cerevisiae: fly L23a can substitute for yeast L25 in ribosome assembly and function

The proposed Drosophila melanogaster L23a ribosomal protein features a conserved C-terminal amino acid signature characteristic of other L23a family members and a unique N-terminal extension [Koyama et al. (Poly(ADP-ribose) polymerase interacts with novel Drosophila ribosomal proteins, L22 and l23a, with unique histone-like amino-terminal extensions. Gene 1999; 226: 339–345)], absent from Saccharomyces cerevisiae L25 that nearly doubles the size of fly L23a. The ability of fly L23a to replace the role of yeast L25 in ribosome biogenesis was determined by creating a yeast strain carrying an L25 chromosomal gene disruption and a plasmid-encoded FLAG-tagged L23a gene. Though affected by a reduced growth rate, the strain is dependent on fly L23a-FLAG function for survival and growth, demonstrating functional compatibility between the fly and yeast proteins. Pulse-chase experiments reveal a delay in rRNA processing kinetics, most notably at a late cleavage step that converts precursor 27S rRNA into mature 25S rRNA, likely contributing to the strain's slower growth pattern. Yet, given the essential requirement for L23(a)/L25 in ribosome biogenesis, there is a remarkable tolerance for accommodating the fly L23a N-terminal extension within the structure of the yeast ribosome. A search of available databases shows that the unique N-terminal extension is shared by multiple insect lineages. An evolutionary perspective on L23a structure and function within insect lineages is discussed.     

A computational investigation on the connection between dynamics properties of ribosomal proteins and ribosome assembly

Assembly of the ribosome from its protein and RNA constituents has been studied extensively over the past 50 years, and experimental evidence suggests that prokaryotic ribosomal proteins undergo conformational changes during assembly. However, to date, no studies have attempted to elucidate these conformational changes. The present work utilizes computational methods to analyze protein dynamics and to investigate the linkage between dynamics and binding of these proteins during the assembly of the ribosome. Ribosomal proteins are known to be positively charged and we find the percentage of positive residues in r-proteins to be about twice that of the average protein: Lys+Arg is 18.7% for E. coli and 21.2% for T. thermophilus. Also, positive residues constitute a large proportion of RNA contacting residues: 39% for E. coli and 46% for T. thermophilus. This affirms the known importance of charge-charge interactions in the assembly of the ribosome. We studied the dynamics of three primary proteins from E. coli and T. thermophilus 30S subunits that bind early in the assembly (S15, S17, and S20) with atomic molecular dynamic simulations, followed by a study of all r-proteins using elastic network models. Molecular dynamics simulations show that solvent-exposed proteins (S15 and S17) tend to adopt more stable solution conformations than an RNA-embedded protein (S20). We also find protein residues that contact the 16S rRNA are generally more mobile in comparison with the other residues. This is because there is a larger proportion of contacting residues located in flexible loop regions. By the use of elastic network models, which are computationally more efficient, we show that this trend holds for most of the 30S r-proteins.     

Saccharomyces cerevisiae ribosomal protein L26 is not essential for ribosome assembly and function

Ribosomal proteins play important roles in ribosome biogenesis and function. Here, we study the evolutionarily conserved L26 in Saccharomyces cerevisiae, which assembles into pre-60S ribosomal particles in the nucle(ol)us. Yeast L26 is one of the many ribosomal proteins encoded by two functional genes. We have disrupted both genes; surprisingly, the growth of the resulting rpl26 null mutant is apparently identical to that of the isogenic wild-type strain. The absence of L26 minimally alters 60S ribosomal subunit biogenesis. Polysome analysis revealed the appearance of half-mers. Analysis of pre-rRNA processing indicated that L26 is mainly required to optimize 27S pre-rRNA maturation, without which the release of pre-60S particles from the nucle(ol)us is partially impaired. Ribosomes lacking L26 exhibit differential reactivity to dimethylsulfate in domain I of 25S/5.8S rRNAs but apparently are able to support translation in vivo with wild-type accuracy. The bacterial homologue of yeast L26, L24, is a primary rRNA binding protein required for 50S ribosomal subunit assembly in vitro and in vivo. Our results underscore potential differences between prokaryotic and eukaryotic ribosome assembly. We discuss the reasons why yeast L26 plays such an apparently nonessential role in the cell.     

Interrelationships between yeast ribosomal protein assembly events and transient ribosome biogenesis factors interactions in early pre-ribosomes

Early steps of eukaryotic ribosome biogenesis require a large set of ribosome biogenesis factors which transiently interact with nascent rRNA precursors (pre-rRNA). Most likely, concomitant with that initial contacts between ribosomal proteins (r-proteins) and ribosome precursors (pre-ribosomes) are established which are converted into robust interactions between pre-rRNA and r-proteins during the course of ribosome maturation. Here we analysed the interrelationship between r-protein assembly events and the transient interactions of ribosome biogenesis factors with early pre-ribosomal intermediates termed 90S pre-ribosomes or small ribosomal subunit (SSU) processome in yeast cells. We observed that components of the SSU processome UTP-A and UTP-B sub-modules were recruited to early pre-ribosomes independently of all tested r-proteins. On the other hand, groups of SSU processome components were identified whose association with early pre-ribosomes was affected by specific r-protein assembly events in the head-platform interface of the SSU. One of these components, Noc4p, appeared to be itself required for robust incorporation of r-proteins into the SSU head domain. Altogether, the data reveal an emerging network of specific interrelationships between local r-protein assembly events and the functional interactions of SSU processome components with early pre-ribosomes. They point towards some of these components being transient primary pre-rRNA in vivo binders and towards a role for others in coordinating the assembly of major SSU domains.     

Isolation of yeast ribosomal proteins L3 and L2 for immunological studies

We report on a rapid method for the isolation and purification of the yeast ribosomal proteins L3 and L2 using a simple instrumentation. Preparative dodecyl sulfate polyacrylamide gel electrophoresis was applied to the separation of cytoplasmatic ribosomal proteins of the large subunit from the yeast Saccharomyces cerevisiae. The polypeptides were removed from gel slices by electrophoretic elution. Subsequent analytical electrophoresis showed groups of proteins in all but two fractions. The latter were further analysed by a two-dimensional gel electrophoresis system which disclosed the purity of two polypeptides. They were identified as L3 and L2. Their molecular masses were 51.5 and 44 kDa as estimated from the gels. A possible application to the isolation of other yeast ribosomal proteins is discussed. An antiserum against the polypeptide L3 was raised in a rabbit. Applying an enzyme-linked immunosorbent assay (ELISA) we were able to determine the relative antibody concentration. Its specificity was demonstrated by immunoblotting.     

Ribosomal proteins L15 and L16 are mere late assembly proteins of the large ribosomal subunit. Analysis of an Escherichia coli mutant lacking L15

The (minus L15) character from the Escherichia coli strain AM16.98 was transduced to an RNase-deficient strain in order to enable a reconstitution analysis. The following results were obtained. 1) The strain lacking L15 showed a 2-3-fold prolonged generation time and the 70 S ribosomes a reduced tendency toward dissociation. 2) Active particles could not be reconstituted unless L15 was added. Addition of L15 regained activity, even if L15 was added after the two-step procedure during a third incubation. However, a modification of the standard two-step reconstitution procedure (lowering NH4+ from 400 to 240 mM and the incubation temperature of the second step from 50 to 47 degrees C) yielded 100% active particles in the absence of L15. Active particles could be formed which even lacked L15, L16, and L30. Addition of either L15 or L16 accelerated the formation of active particles in the second step by a factor of five, and both proteins together by a factor of more than 20. 3) The activation energy of the rate-limiting step of the second incubation was surprisingly reduced for about 20 kcal/mol in the absence of L15, although the corresponding rates were two to five times slower. We conclude 1) that L15 and L16 are late assembly proteins which accelerate the formation of active particles during the late assembly but are neither needed for the early assembly nor essential for ribosomal functions; 2) that some routes of the late assembly (e.g. incorporation of L16) are changing their significance depending on the NH4+ concentration and the absence and presence of L15; and 3) that different reactions are rate limiting during the second step incubation in the presence and absence of L15, respectively, and that the corresponding reaction rates exhibit a different temperature dependence.     

Arginine methylation of ribosomal protein S3 affects ribosome assembly

The human ribosomal protein S3 (rpS3), a component of the 40S small subunit in the ribosome, is a known multi-functional protein with roles in DNA repair and apoptosis. We recently found that the arginine residue(s) of rpS3 are methylated by protein arginine methyltransferase 1 (PRMT1). In this paper, we confirmed the arginine methylation of rpS3 protein both in vitro and in vivo. The sites of arginine methylation are located at amino acids 64, 65 and 67. However, mutant rpS3 (3RA), which cannot be methylated at these sites, cannot be transported into the nucleolus and subsequently incorporated into the ribosome. Our results clearly show that arginine methylation of rpS3 plays a critical role in its import into the nucleolus, as well as in small subunit assembly of the ribosome.     

Deletion of L4 domains reveals insights into the importance of ribosomal protein extensions in eukaryotic ribosome assembly

Numerous ribosomal proteins have a striking bipartite architecture: a globular body positioned on the ribosomal exterior and an internal loop buried deep into the rRNA core. In eukaryotes, a significant number of conserved r-proteins have evolved extra amino- or carboxy-terminal tail sequences, which thread across the solvent-exposed surface. The biological importance of these extended domains remains to be established. In this study, we have investigated the universally conserved internal loop and the eukaryote-specific extensions of yeast L4. We show that in contrast to findings with bacterial L4, deleting the internal loop of yeast L4 causes severely impaired growth and reduced levels of large ribosomal subunits. We further report that while depleting the entire L4 protein blocks early assembly steps in yeast, deletion of only its extended internal loop affects later steps in assembly, revealing a second role for L4 during ribosome biogenesis. Surprisingly, deletion of the entire eukaryote-specific carboxy-terminal tail of L4 has no effect on viability, production of 60S subunits, or translation. These unexpected observations provide impetus to further investigate the functions of ribosomal protein extensions, especially eukaryote-specific examples, in ribosome assembly and function.     

An acidic amino acid cluster regulates the nucleolar localization and ribosome assembly of human ribosomal protein L22

The control of human ribosomal protein L22 (rpL22) to enter into the nucleolus and its ability to be assembled into the ribosome is regulated by its sequence. The nuclear import of rpL22 depends on a classical nuclear localization signal of four lysines at positions 13-16. RpL22 normally enters the nucleolus via a compulsory sequence of KKYLKK (I-domain, positions 88-93). An acidic residue cluster at the C-terminal end (C-domain) plays a nuclear retention role. The retention is concealed by the N-domain (positions 1-9) which weakly interacts with the C-domain as demonstrated in the yeast two-hybrid system. Once it reaches the nucleolus, the question of whether rpL22 is assembled into the ribosome depends upon the presence of the N-domain. This suggests that the N-domain, on dissociation from its interaction with the C-domain, binds to a specific region of the 28S rRNA for ribosome assembly.     

Mapping the essential structures of human ribosomal protein L7 for nuclear entry, ribosome assembly and function

Human large subunit protein L7 carries multiple nuclear localization signals (NLS) in its structure: there are three monobasic partite NLSs at the NH2-region of the first 54 amino acid residues and a bipartite in the middle section at position of 156-167. The C-region of the last 50 amino acid residues displays membrane binding nature, and might involve in forming a nuclear microbody for pre-nucleolar ribosome assembly. The middle section covers 144 amino acid residues which are essential for the structure and function of ribosome. This is evident from findings that truncated L7 without the NH2-region or the C-region, or missing both regions, is capable of reaching nucleolus and incorporating in ribosome, however, only ribosomes bearing truncated L7 without the NH2-region is capable of engaging in polysome formation. Combining with the phylogenic findings from homologous sequence alignment, the NH2-region of L7, besides being as a eukaryotic expansion segment, can be excluded from building a functional eukaryotic ribosome.     

The primary structure of rat liver ribosomal protein L37. Homology with yeast and bacterial ribosomal proteins

The covalent structure of the rat liver 60 S ribosomal subunit protein L37 was determined. Twenty-four tryptic peptides were purified and the sequence of each was established; they accounted for all 111 residues of L37. The sequence of the first 30 residues of L37, obtained previously by automated Edman degradation of the intact protein, provided the alignment of the first 9 tryptic peptides. Three peptides (CN1, CN2, and CN3) were produced by cleavage of protein L37 with cyanogen bromide. The sequence of CN1 (65 residues) was established from the sequence of secondary peptides resulting from cleavage with trypsin and chymotrypsin. The sequence of CN1 in turn served to order tryptic peptides 1 through 14. The sequence of CN2 (15 residues) was determined entirely by a micromanual procedure and allowed the alignment of tryptic peptides 14 through 18. The sequence of the NH2-terminal 28 amino acids of CN3 (31 residues) was determined; in addition the complete sequences of the secondary tryptic and chymotryptic peptides were done. The sequence of CN3 provided the order of tryptic peptides 18 through 24. Thus the sequence of the three cyanogen bromide peptides also accounted for the 111 residues of protein L37. The carboxyl-terminal amino acids were identified after carboxypeptidase A treatment. There is a disulfide bridge between half-cystinyl residues at positions 40 and 69. Rat liver ribosomal protein L37 is homologous with yeast YP55 and with Escherichia coli L34. Moreover, there is a segment of 17 residues in rat L37 that occurs, albeit with modifications, in yeast YP55 and in E. coli S4, L20, and L34.