Role of protein L25 and its contact with protein L16 in maintaining the active state of <Emphasis Type="Italic">Escherichia coli</Emphasis> ribosomes <Emphasis Type="Italic">in vivo</Emphasis>

A ribosomal protein of the L25 family specifically binding to 5S rRNA is an evolutionary feature of bacteria. Structural studies showed that within the ribosome this protein contacts not only 5S rRNA, but also the C-terminal region of protein L16. Earlier we demonstrated that ribosomes from the ΔL25 strain of Escherichia coli have reduced functional activity. In the present work, it is established that the reason for this is a fraction of functionally inactive 50S ribosomal subunits. These subunits have a deficit of protein L16 and associate very weakly with 30S subunits. To study the role of the contact of these two proteins in the formation of the active ribosome, we created a number of E. coli strains containing protein L16 with changes in its C-terminal region. We found that some mutations (K133L or K127L/K133L) in this protein lead to a noticeable slowing of cell growth and decrease in the activity of their translational apparatus. As in the case of the ribosomes from the ΔL25 strain, the fraction of 50S subunits, which are deficient in protein L16, is present in the ribosomes of the mutant strains. All these data indicate that the contact with protein L25 is important for the retention of protein L16 within the E. coli ribosome in vivo. In the light of these findings, the role of the protein of the L25 family in maintaining the active state of the bacterial ribosome is discussed.     

Interaction of the cytoplasmic membrane and ribosomes in Escherichia coli; altered ribosomal proteins in sucrose-dependent spectinomycin-resistant mutants.

Alterations in the ribosomes of sucrose-dependent spectinomycin-resistant (Sucd-Spcr) mutants of Escherichia coli were studied. Subunit exchange experiments showed that 30S subunits were responsible for the resistance of ribosomes to spectinomycin in all Sucd-Spcr mutants tested. Proteins of 30S ribosomes were analyzed by carboxymethyl cellulose column chromatography based on their elution positions. Mutants YM22 and YM93 had an altered 30S ribosomal protein component, S5, and mutant YM50 had an altered protein, S4. Although a shift of elution position was not detected for all the 30S ribosomal proteins from mutant YM101, the amount of protein S3 was appreciably lowered in the isolated 30S subunits. A partial reconstitution experiment with protein S3 prepared from both the wild-type strain and YM101 revealed that the mutant had altered protein S3 which is responsible for the spectinomycin resistance. These alterations in 30S subunits are discussed in relation to the interaction between ribosomes and the cytoplasmic membrane.     

A second-site mutation at glutamate-257 that restores the function of the mutant yeast ribosomal protein L5 containing lysine-270,271-->arginine

A genetic approach was used to identify interacting regions of yeast ribosomal protein L5 (also known as L1, L1a, or YL3). Previous studies from our laboratory showed that residues K270 and K271 in protein L5 are essential for its function. The mutant L5 protein in which both residues were replaced by arginine residues (K270,271R) exhibited about 80% RNA binding capability compared to the wild-type and the mutant protein was assembled into the 60S ribosomal subunits in vivo. The yeast strain expressing this mutant protein in a homozygous form was lethal (Biochim. Biophys. Acta 1308 (1996) 133-141). In the present study, this non-functional mutant was used to select intragenic suppressors. A spontaneous, intragenic suppressor which contained an E257K substitution (in addition to the primary mutations) was identified. The suppressor protein bound about 60% of yeast 5S rRNA in vitro compared to the wild-type. To gain more insight into the nature of the intragenic suppressor, additional mutant proteins in which E257 was substituted by a variety of amino acids were produced by site-directed mutagenesis. The ability of each mutant protein to bind yeast 5S rRNA in vitro and to suppress the lethal effect of the double K270,271 mutation in vivo were examined. Results suggest communication between two non-contiguous domains on protein L5 and that several factors, such as electrostatic interaction and hydrogen bonding are likely to play a role in this global communication. Mutation studies on E257 alone also reveal that substitutions of this residue in L5 protein could affect cell growth under specified conditions, but a variety of changes could be tolerated without serious deleterious effects. We propose a working model in which E257 is located in a loop and the dynamic as well as the flexibility of this loop is important for L5 function.     

A 3' splice site mutation in a nuclear gene encoding a mitochondrial ribosomal protein in Neurospora crassa

We showed previously that the cyt-21+ gene of Neurospora crassa encodes a mitochondrial ribosomal protein homologous to Escherichia coli ribosomal protein S-16 (Kuiper, M. T. R., Akins, R. A., Holtrop, M., de Vries, H., and Lambowitz, A. M. (1988) J. Biol. Chem. 263, 2840-2847). A mutation in this gene, cyt-21-1, results in deficiency of mitochondrial small ribosomal subunits and small rRNA (Collins, R. A., Bertrand, H., LaPolla, R. J., and Lambowitz, A. M. (1979) Mol. Gen. Genet. 177, 73-84). In the present work, cloning and sequencing of the cyt-21-1 mutant allele show that it contains a single dG to dA transition at the 3' splice site AG of the first intron in the protein coding region. This mutation leads to inactivation of the normal 3' splice site and activation of a cryptic 3' splice site, 15 nucleotides downstream. The use of this cryptic splice site results in an in-frame deletion of 5 amino acids from the cyt-21 protein. Comparison of mutant and wild-type mitochondrial small ribosomal subunit proteins showed one protein, S-24, with an altered electrophoretic mobility, consistent with the predicted deletion. The mutant ribosomal protein is still capable of binding to mitochondrial small ribosomal subunits, but results in abnormal mitochondrial ribosome assembly.     

The C-terminal domain of dnaQ contains the polymerase binding site

The Escherichia coli dnaQ gene encodes the 3'-->5' exonucleolytic proofreading (epsilon) subunit of DNA polymerase III (Pol III). Genetic analysis of dnaQ mutants has suggested that epsilon might consist of two domains, an N-terminal domain containing the exonuclease and a C-terminal domain essential for binding the polymerase (alpha) subunit. We have created truncated forms of dnaQ resulting in epsilon subunits that contain either the N-terminal or the C-terminal domain. Using the yeast two-hybrid system, we analyzed the interactions of the single-domain epsilon subunits with the alpha and theta subunits of the Pol III core. The DnaQ991 protein, consisting of the N-terminal 186 amino acids, was defective in binding to the alpha subunit while retaining normal binding to the theta subunit. In contrast, the NDelta186 protein, consisting of the C-terminal 57 amino acids, exhibited normal binding to the alpha subunit but was defective in binding to the theta subunit. A strain carrying the dnaQ991 allele exhibited a strong, recessive mutator phenotype, as expected from a defective alpha binding mutant. The data are consistent with the existence of two functional domains in epsilon, with the C-terminal domain responsible for polymerase binding.     

Amiloride resistance in the methanoarcheonMethanothermobacter thermoautotrophicus: Characterization of membrane-associated proteins

An amiloride-resistant mutant with diminished Na+/H+ antiporter activity was isolated from Methanothermobacter thermoautotrophicus. To define the protein basis of amiloride resistance, the composition of membrane-associated proteins was partially characterized and compared with that of the wild type strain. An abundant 670-kDa membrane-associated protein that was present only in the mutant strain was analyzed by MALDI-TOF MS and identified as a coenzyme F420-reducing hydrogenase. The amiloride resistance was not accompanied by changes in protein size or changes in the level of subunits A or B of the A1A0-type ATP synthase; on the other hand, the SDS-PAGE patterns of the chloroform-methanol extract of membranes from both strains were different. Two bands with calculated molecular mass 16 and 11 kDa were identified as MtrD and AtpK, respectively. The observed over-expression of a 22.7-kDa protein in the mutant cells may represent the multimeric form of the MtrD subunit. These results show that the impairment of the Na+/H+ antiporter system in the amiloride-resistant mutant of Methanothermobacter thermoautotrophicus is accompanied by only small changes in a few membrane-associated proteins.     

The requirement for eukaryotic initiation factor 4A (elF4A) in translation is in direct proportion to the degree of mRNA 5' secondary structure

Eukaryotic initiation factor (elF) 4A functions as a subunit of the initiation factor complex elF4F, which mediates the binding of mRNA to the ribosome. elF4A possesses ATPase and RNA helicase activities and is the prototype for a large family of putative RNA helicases (the DEAD box family). It is thought that the function of elF4A during translation initiation is to unwind the mRNA secondary structure in the 5' UTR to facilitate ribosome binding. However, the evidence to support this hypothesis is rather indirect, and it was reported that elF4A is also required for the translation of mRNAs possessing minimal 5' UTR secondary structure. Were this hypothesis correct, the requirement for elF4A should correlate with the degree of mRNA secondary structure. To test this hypothesis, the effect of a dominant-negative mutant of mammalian elF4A on translation of mRNAs with various degrees of secondary structure was studied in vitro. Here, we show that mRNAs containing stable secondary structure in the 5' untranslated region are more susceptible to inhibition by the elF4A mutant. The mutant protein also strongly inhibits translation from several picornavirus internal ribosome entry sites (IRES), although to different extents. UV crosslinking of elF4F subunits and elF4B to the mRNA cap structure is dramatically reduced by the elF4A mutant and RNA secondary structure. Finally, the elF4A mutant forms a more stable complex with elF4G, as compared to the wild-type elF4A, thus explaining the mechanism by which substoichiometric amounts of mutant elF4A inhibit translation.     

Suppression of defective ribosome assembly in a rbfA deletion mutant by overexpression of Era,an essential GTPase in Escherichia coli

Era is a small GTP-binding protein and essential for cell growth in Escherichia coli. It consists of two domains: N-terminal GTP-binding and C-terminal RNA-binding KH domains. It has been shown to bind to 16S rRNAs and 30S ribosomal subunits in vitro. Here, we report that a precursor of 16S rRNA accumulates in Era-depleted cells. The accumulation of the precursors is also seen in a cold-sensitive mutant, E200K, in which the mutation site is located in the C-terminal domain. The major precursor molecule accumulated seems to be 17S rRNA, containing extra sequences at both 5' and 3' ends of 16S rRNA. Moreover, the amounts of both 30S and 50S ribosomal subunits relative to the amount of 70S monosomes increase in Era-depleted and E200K mutant cells. The C-terminal KH domain has a high structural similarity to the RbfA protein, a cold shock protein that also specifically associates with 30S ribosomal subunits. RbfA is essential for cell growth at low temperature, and a precursor of 16S rRNA accumulates in an rbfA deletion strain. The 16S rRNA precursor seems to be identical in size to that accumulated in Era mutant cells. Surprisingly, the cold-sensitive cell growth of the rbfA deletion cells was partially suppressed by overproduction of the wild-type Era. The C-terminal domain alone was not able to suppress the cold-sensitive phenotype, whereas Era-dE, which has a 10-residue deletion in a putative effector region of the N-terminal domain, functioned as a more efficient suppressor than the wild-type Era. It was found that Era-dE suppressed defective 16S rRNA maturation, resuming a normal polysome profile to reduce highly accumulated free 30S and 50S subunits in the rbfA deletion cells. These results indicate that Era is involved in 16S rRNA maturation and ribosome assembly.     

Analysis of the structures of the subunits of the cytochrome bc1 complex from beef heart mitochondria

The interaction of the protein subunits of the bc1 complex from beef heart is analysed on the basis of protein chemical data and of secondary structure predictions suggesting a large number of amphipathic helices. Electrostatic interactions, i.e. helix-dipole interactions and ionic bonds, may play a major role in the stabilisation of the arrangement of the subunits within the multi-protein complex, formation of subcomplexes and maintenance of the steric strain of cytochrome b. A model of the heme-carrying 'core' of cytochrome b, i.e. of helices II-V, is presented consisting of a twisted '4-alpha-helical' bundle held together by helix-dipole interactions and stabilised by the interaction with other protein subunits of the bc1 complex.     

Construction and functional analysis of ribosomal 5S RNA from Escherichia coli with single base changes in the ribosomal protein binding sites

The ribosomal 5S RNA gene from E. coli was altered by oligonucleotide-directed mutagenesis at positions A66 and U103. The mutant genes were cloned into an expression vector and selectively transcribed in an UV-sensitive E. coli strain using a modified maxicell system. The mutant 5S RNA genes were found to be transcribed and processed normally. The 5S RNA molecules were assembled into 50S ribosomal subunits. Under in vitro conditions the stability of the mutant 70S ribosomes seemed, however, to be reduced, since they dissociated into their subunits more easily than those of the wild type. The isolated mutated 5S RNAs with base changes in the ribosomal protein binding sites for L18 and L25, together with a point mutant at G41 (G to C), constructed earlier, were tested for their capacity to bind the 5S RNA binding proteins L5, L18 and L25. The following effects were observed: The base change A66 to C within the L18 binding site did not affect the binding of the ribosomal protein L18 but enhanced the stability of the L25-5S RNA complex considerably. The base changes U103 to G and G41 to C slightly reduced the binding of L5 and L25 whereas the binding of L18 to the mutant 5S RNAs was not altered. In addition 70S ribosomes with the single point mutations in their 5S RNAs were tested in their tRNA binding capacity. Mutants containing a C41 in their 5S RNA showed a reduction in the poly(U)-dependent Phe-tRNA binding, whereas the mutations to C66 and G 103 lead to completely inactive ribosomes in the same assay. Based on previous results a spatial model of the 5S RNA molecule is presented which is consistent with the findings reported in this paper.     

Homologous mutations on different subunits cause unequal but additive effects on n-alcohol block in the nicotinic receptor pore.

Hydrophobic antagonists of the nicotinic acetylcholine receptor inhibit channel activity by binding within the transmembrane pore formed by the second of four transmembrane domains (M2) on each of the receptor's subunits. Hydrophobic mutagenesis near the middle (10' locus) of the alpha-subunit M2 domain results in channels that are much more sensitive to block by long-chain alcohols and general anesthetics, indicating that the inhibitory site on wild-type receptors is nearby. To determine whether other receptor subunits also contribute to the blocker site, the hydrophobic mutagenesis strategy was extended to all four subunits at 10' loci. alpha S10'l causes the largest increase in apparent hexanol binding (4.3-fold compared to wild type), approximately twice the size of the change caused by beta T10'l (2.2-fold). gamma A10'l and delta A10'l mutations cause much smaller changes in apparent hexanol binding affinity (about 1.2-fold each), even when corrected for their smaller degree of side-chain hydrophobicity changes. When 10'l mutant subunits are coexpressed, the change from wild type in apparent hexanol binding energy (delta delta Gmixture) is roughly equal to the sum of hexanol binding energy changes for the constituent mutant subunits (sigma delta delta Gsubunits). The simplest model consistent with these results is one in which hydrophobic blockers make simultaneous contact with all five M2 10' residues, but the extent of contact is much greater for the alpha and beta than for gamma and delta side chains.     

Identification of the dicyclohexylcarbodiimide-reactive protein component of the adenosine 5''-triphosphate energy-transducing system of Escherichia coli.

Membranes of Escherichia coli contain an adenosine 5'-triphosphate (ATP) energy-transducing system that is inhibited by treatment with dicyclohexylcarbodiimide (DCCD). The carbodiimide-reactive protein component of this system has been identified after treatment with [14C]DCCD. This protein has an apparent molecular weight of 9,000 as judged from acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and is extracted from the membrane with chloroform-methanol (2:1). These properties are similar to the analogous protein previously identified in mitochondria (Cattell et al., 1971). A mutant strain, RF-7, has been isolated which derives energy from oxidative phosphorylation in the presence of 5 mM DCCD. The ATP hydrolase activity of the membraned system in the mutant was considerably less sensitive to inhibition by DCCD than that in the wild type. The carbodiimide-reactive protein, which was easily labeled by [14C]DCCD in the wild type, was labeled much less rapidly in the carbodiimide-resistant mutant. It is thus concluded that the reaction of DCCD with this specific protein leads to inhibition of the ATP energy-transducing reactions. The mutation causing carbodiimide resistance in strain RF-7 was mapped. It is cotransduced with the uncA gene at a frequency exceeding 90%. The mutationally altered protein causing the carbodiimide resistance was not conclusively identified. However, reconstitution experiments indicate that the altered protein is not one of the subunits of the soluble ATP hydrolase activity, which can be removed from the membrane by washing with 1 mM tris(hydroxymethyl)aminomethane buffer lacking Mg2+. The carbodiimide-reactive protein remains with the membrane residue after removal of the soluble ATP hydrolase and is thus distinct from these subunits as well.     

Identification of a region of Escherichia coli ribosomal protein L2 required for the assembly of L16 into the 50 S ribosomal subunit

In vitro mutagenesis of rplB was used to generate changes in a conserved region of Escherichia coli ribosomal protein L2 between Gly221 and His231. Mutants were selected by temperature sensitivity using an inducible expression system. A mutant L2 protein with the deletion of Thr222 to Asp228 was readily distinguishable from wild-type L2 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and ribosomes from the strain overexpressing this mutant protein were characterized by sucrose density gradient centrifugation and protein composition. In addition to 30 S and 50 S ribosomal subunits, cell lysates contained a new component that sedimented at 40 S in 1 mM Mg2+ and at 48 S in 10 mM Mg2+. These particles contained mutant L2 protein exclusively, completely lacked L16, and had reduced amounts of L28, L33, and L34. They did not reassociate with 30 S ribosomal subunits and were inactive in polyphenylalanine synthesis. Other mutants in the same conserved region, including the substitution of His229 by Gln229, produced similar aberrant 50 S particles that sedimented at 40 S and failed to associate with 30 S subunits.     

Dob1p (Mtr4p) is a putative ATP-dependent RNA helicase required for the 3'' end formation of 5.8S rRNA in Saccharomyces cerevisiae.

The temperature-sensitive mutation, dob1-1, was identified in a screen for dependence on overexpression of the yeast translation initiation factor eIF4B (Tif3p). Dob1p is an essential putative ATP-dependent RNA helicase. Polysome analyses revealed an under accumulation of 60S ribosomal subunits in the dob1-1 mutant. Pulse-chase labelling of pre-rRNA showed that this was due to a defect in the synthesis of the 5.8S and 25S rRNAs. Northern and primer extension analyses in the dob1-1 mutant, or in a strain genetically depleted of Dob1p, revealed a specific inhibition of the 3' processing of the 5.8S rRNA from its 7S precursor. This processing recently has been attributed to the activity of the exosome, a complex of 3'-->5' exonucleases that includes Rrp4p. In vivo depletion of Dob1p also inhibits degradation of the 5' external transcribed spacer region of the pre-rRNA. A similar phenotype was observed in rrp4 mutant strains and, moreover, the dob1-1 and rrp4-1 mutations show a strong synergistic growth inhibition. We propose that Dob1p functions as a cofactor for the exosome complex that unwinds secondary structures in the pre-rRNA that otherwise block the progression of the 3'-->5' exonucleases.     

Pta1, a Component of Yeast CF II, Is Required for Both Cleavage and Poly(A) Addition of mRNA Precursor

CF II, a factor required for cleavage of the 3' ends of mRNA precursor in Saccharomyces cerevisiae, has been shown to contain four polypeptides. The three largest subunits, Cft1/Yhh1, Cft2/Ydh1, and Brr5/Ysh1, are homologs of the three largest subunits of mammalian cleavage-polyadenylation specificity factor (CPSF), an activity needed for both cleavage and poly(A) addition. In this report, we show by protein sequencing and immunoreactivity that the fourth subunit of CF II is Pta1, an essential 90-kDa protein originally implicated in tRNA splicing. Yth1, the yeast homolog of the CPSF 30-kDa subunit, is not detected in this complex. Extracts prepared from pta1 mutant strains are impaired in the cleavage and the poly(A) addition of both GAL7 and CYC1 substrates and exhibit little processing activity even after prolonged incubation. However, activity is efficiently rescued by the addition of purified CF II to the defective extracts. Extract from a strain with a mutation in the CF IA subunit Rna14 also restored processing, but extract from a brr5-1 strain did not. The amounts of Pta1 and other CF II subunits are reduced in pta1 strains, suggesting that levels of the subunits may be coordinately regulated. Coimmunoprecipitation experiments indicate that the CF II in extract can be found in a stable complex containing Pap1, CF II, and the Fip1 and Yth1 subunits of polyadenylation factor I. While purified CF II does not appear to retain the association with these other factors, this larger complex may be the form recruited onto pre-mRNA in vivo. The involvement of Pta1 in both steps of mRNA 3'-end formation supports the conclusion that CF II is the functional homolog of CPSF.