Functional requirement of a site-specific ribose methylation in ribosomal RNA.

The product of the PET56 nuclear gene of Saccharomyces cerevisiae was shown to be required for ribose methylation at a universally conserved nucleotide in the peptidyl transferase center of the mitochondrial large ribosomal RNA (21S rRNA). Cells reduced in this activity were deficient in formation of functional large subunits of the mitochondrial ribosome. The purified Pet56 protein catalyzed the site-specific formation of 2'-O-methylguanosine on in vitro transcripts of both mitochondrial 21S rRNA and Escherichia coli 23S rRNA. These results provide evidence for an essential modified nucleotide in rRNA.

Chemical sequence control of beta-sheet assembly in macromolecular crystals of periodic polypeptides.

A family of uniform periodic polypeptides has been prepared by bacterial expression of the corresponding artificial genes, with the objective of exploring the potential for control of supramolecular organization in genetically engineered protein-based polymeric materials. The repeating units of the polypeptides consist of oligomeric alanyl-glycine sequences interspersed with glutamic acid residues inserted at intervals of 8 to 14 amino acids. Crystallization of such materials from formic acid produces beta-sheet structures in the solid state, as shown by vibrational spectroscopy, nuclear magnetic resonance spectroscopy, and wide-angle x-ray diffraction. The diffraction results, together with observations from electron microscopy, are consistent with the formation of needle-shaped lamellar crystals whose thickness is controlled by the periodicity of the primary sequence. These results can be used to control solid-state structure in macromolecular materials.

Structure and regulation of a nuclear gene in Saccharomyces cerevisiae that specifies MRP7, a protein of the large subunit of the mitochondrial ribosome.

The gene for MRP7, a 40-kilodalton protein of the large subunit of the yeast mitochondrial ribosome, was identified in a lambda gt11 expression library by immunological screening with a monoclonal antibody to MRP7. An intact copy of MRP7 was then isolated from a yeast genomic library by colony hybridization. Gene disruption showed that MRP7 protein was essential for ribosomal function. Sequencing of MRP7 revealed a coding region for a basic (pI 10.6), 43.2-kilodalton protein containing 371 amino acid residues. Amino acid residues 28 to 112 of the deduced MRP7 sequence aligned with the 84 residues of the Escherichia coli ribosomal protein L27, but no significant similarity was detected between the carboxy-terminal 259 amino acids of MRP7 and other protein sequences in existing computer data bases. Within the aligned region, there was 49% amino acid identity between MRP7 and L27, compared with the 57% identity observed between L27 and its homolog in Bacillus stearothermophilus. The steady-state levels of the MRP7 protein and its mRNA were monitored in response to catabolite repression and to increased dosage of the MRP7 gene. The response to catabolite repression was characterized by a ninefold change in the level of the protein and little, if any, change in the level of the mRNA. In cells carrying the MRP7 gene on a high-copy-number plasmid, the mRNA was increased 20-fold, but there was no significant increase in MRP7 protein. Furthermore, MRP7 mRNA and protein accumulated at normal levels in [rho0] cells, which are devoid of 21S rRNA, indicating that the protein is relatively stable in the absence of ribosome assembly. Together, these results suggest that MRP7 is regulated posttranscriptionally, probably at the level of protein synthesis rather than protein turnover.

Structure and regulation of a nuclear gene in Saccharomyces cerevisiae that specifies MRP13, a protein of the small subunit of the mitochondrial ribosome.

MRP13 is defined by biochemical criteria as a 35-kilodalton small subunit protein of the yeast mitochondrial ribosome. The MRP13 gene was identified by immunological screening of a yeast genomic library in lambda gt11 and a functional copy of the gene has been cloned on a 2.2-kilobase BglII fragment. Sequencing of this fragment showed that the MRP13 coding region specifies a 324-amino-acid basic protein with a calculated Mr of 37,366. Computer searches failed to reveal any significant sequence similarity to previously identified ribosomal proteins or to the sequences in the current National Biomedical Research Foundation data base. Cells carrying disrupted copies of MRP13 lacked the MRP13 protein but were not impaired in either mitochondrial protein synthesis or assembly of 37S ribosomal subunits, indicating that, like L29 and L30 in Escherichia coli (M. Lotti, E. R. Dabbs, R. Hasenbank, M. Stöffler-Meilicke, and G. Stöffler, Mol. Gen. Genet. 192:295-300, 1983), MRP13 is not essential for ribosome synthesis or function. Analysis of the sequence in the MRP13 5'-flanking region revealed the closely linked gene for the cytoplasmic ribosomal protein rp39A. The rp39A coding region began at nucleotide -846 and ended at -325 with respect to the MRP13 translational start. The steady-state levels of the MRP13 mRNA were determined in response to carbon catabolite repression, variation in the mitochondrial genetic background, and increased gene dosage of MRP13. In [rho+] cells, transcript levels were repressed severalfold by growth in glucose compared with growth in either galactose or nonfermentable carbon sources. In respiratory-deficient strains ([rho0], [mit-]), however, transcription appeared to be largely derepressed even in the presence of high concentrations of glucose. Despite high levels of the MRP13 transcripts in [rho0] cells, the MRP13 protein did not accumulate, suggesting that the protein is relatively unstable in the absence of ribosome assembly. Cells carrying the MRP13 gene on a multiple-copy plasmid overproduced the mRNA in rough proportion to the gene dosage and the protein in a significant but lesser amount. The results indicate that MRP13 expression is regulated predominantly at the transcriptional level in response to catabolite repression and the cellular capacity for respiration and, in addition, that protein levels appear to be modulated posttranscriptionally by degradation of free copies of the MRP13 protein.

T7 RNA polymerase-dependent expression of COXII in yeast mitochondria.

An in vivo expression system has been developed for controlling the transcription of individual genes in the mitochondrial genome of Saccharomyces cerevisiae. The bacteriophage T7 RNA polymerase (T7Pol), fused to the COXIV mitchondrial import peptide and expressed under the control of either the GAL1 or the ADH1 promoter, efficiently transcribes a target gene, T7-COX2, in the mitochondrial genome. Cells bearing the T7-COX2 gene, but lacking wild-type COX2, require T7Pol for respiration. Functional expression of T7-COX2 is completely dependent on the COX2-specific translational activator Pet111p, despite additional nucleotides at the 5' end of the T7-COX2 transcript. Expression of mitochondrion-targeted T7Pol at high levels from the GAL1 promoter has no detectable effect on mitochondrial function in rho+ cells lacking the T7-COX2 target gene, but in cells with T7-COX2 integrated into the mitochondrial genome, an equivalent level of T7Pol expression causes severe respiratory deficiency. In comparison with wild-type COX2 expression, steady-state levels of T7-COX2 mRNA increase fivefold when transcription is driven by T7Pol expressed from the ADH1 promoter, yet COXII protein levels and cellular respiration rates decrease by about 50%. This discoordinate expression of mRNA and protein provides additional evidence for posttranscriptional control of COX2 expression.

HSP78 encodes a yeast mitochondrial heat shock protein in the Clp family of ATP-dependent proteases.

The Saccharomyces cerevisiae nuclear gene for a 78-kDa mitochondrial heat shock protein (hsp78) was identified in a lambda gt11 expression library through immunological screening with an hsp78-specific monoclonal antibody. Sequencing of HSP78 revealed a long open reading frame capable of encoding an 811-amino-acid, 91.3-kDa basic protein with a putative mitochondrial leader sequence and two potential nucleotide-binding sites. Sequence comparisons revealed that hsp78 is a member of the highly conserved family of Clp proteins and is most closely related to the Escherichia coli ClpB protein, which is thought to be an ATPase subunit of an intracellular ATP-dependent protease. The steady-state levels of HSP78 transcripts and protein varied in response to both thermal stress and carbon source with an approximately 30-fold difference between repressed levels in cells growing fermentatively on glucose at 30 degrees C and derepressed levels in heat-shocked cells growing on a nonfermentable carbon source. The response to heat shock is consistent with the presence of a characteristic heat shock regulatory element in the 5'-flanking DNA. Submitochondrial fractionation showed that hsp78 is a soluble protein located in the mitochondrial matrix. Cells carrying disrupted copies of HSP78 lacked hsp78 but were not impaired in respiratory growth at normal and elevated temperatures or in the ability to survive and retain mitochondrial function after thermal stress. The absence of a strong mitochondrial phenotype in hsp78 mutants is comparable to the nonlethal phenotypes of mutations in other Clp genes in bacteria and yeast. HSP78 is the third gene, with SSC1 and HSP60, known to encode a yeast mitochondrial heat shock protein and the second gene, with HSP104, for a yeast ClpB homolog.

A nuclear mutation that post-transcriptionally blocks accumulation of a yeast mitochondrial gene product can be suppressed by a mitochondrial gene rearrangement.

The nuclear amber mutation, pet494-1, specifically blocks the accumulation of the product of the mitochondrial gene oxi2, cytochrome oxidase subunit III. The pet494-1 mutation does not prevent transcription of the mitochondrial gene since RNA--gel blot hybridizations showed that mutant cells contain normal amounts of an oxi2 transcript, indistinguishable in size from wild-type. A mitochondrial mutation that partially suppresses the nuclear mutation was isolated. The "mitochondrial revertant" behaved as though it contained two different mitochondrial DNAs: one rho+, the other rho-. The suppressor mutation is carried on the rho- mitochondrial DNA and is apparently the result of a gene fusion between oxi2 and another mitochondrial gene, oxi3. This gene rearrangement replaced the normal 5'-non-translated sequence of oxi2 with a portion of the open reading frame of the second intron of oxi3. Novel transcripts of the rearranged gene, containing oxi3 sequences upstream from oxi2 were detected in the mitochondrial revertant. The strain accumulated an electrophoretically variant form of cytochrome oxidase subunit III, probably translated from a new initiation codon. The data are consistent with models in which the PET494 protein acts within the mitochondria to specifically promote the translation of the oxi2 messenger RNA.

HTS1 encodes both the cytoplasmic and mitochondrial histidyl-tRNA synthetase of Saccharomyces cerevisiae: mutations alter the specificity of compartmentation.

Genetic and biochemical evidence shows that a single nuclear gene HTS1 encodes both the mitochondrial and cytoplasmic histidyl-tRNA synthetases (Hts). The gene specifies two messages, one with two in-frame ATGs (-60 and +1) and another with only the downstream ATG (+1). We have made a new set of mutations that enables us to express only the mitochondrial or the cytoplasmic form and compared the subcellular distribution of the Hts1 protein in these mutants and wild type, using an antibody that interacts with both the mitochondrial and cytoplasmic Hts1 as well as Hts1::LacZ fusions. Mutations in the upstream ATG (-60) or frameshift mutations in the presequence affect only the mitochondrial enzyme and not the cytoplasmic enzyme. Mutations in the downstream ATG (+1 ATG to ATC) destroy the function of the cytosolic enzyme, but do not affect the function of the mitochondrial enzyme. Overexpression of this construct restores cytoplasmic function. Cells expressing a truncated form of Hts containing a deletion of the first 20 amino-terminal residues (Htsc) produce a functional cytoplasmic enzyme, which does not provide mitochondrial function. Overexpression of this truncated cytoplasmic protein provides mitochondrial function and produces detectable levels of the synthetase in the mitochondrion. These experiments suggest that Hts1 contains two domains that together allow efficient localization of Htsm to the mitochondrion: an amino-terminal presequence in the mitochondrial precursor that is likely cleaved upon delivery to the mitochondrion and a second amino-terminal sequence (residues 21-53) present in both the precursor and the cytoplasmic form. Neither one by itself is sufficient to act as an efficient mitochondrial targeting signal. Using our antibody we have been able to detect a protein of increased molecular mass that corresponds to that of the predicted precursor. Taken together these studies show that the specificity of compartmentation of the Hts protein depends upon both the primary sequence and the concentration of the protein in the cell.

Implications of a functional large ribosomal RNA with only three modified nucleotides.

The sequence and structure of the peptidyl transferase region of large subunit ribosomal RNA is highly conserved and specific modified nucleotides could be important structural or functional elements in the catalytic center responsible for peptide bond formation. In fact, it has not been possible to reconstitute active E coli 50S subunits from in vitro transcripts of 23S rRNA and total 50S proteins. It is significant therefore, that the PET56 gene of yeast encodes an essential ribose methyltransferase that specifically modifies a universally conserved nucleotide, G2270, in the peptidyl transferase center of the mitochondrial large ribosomal RNA (21S). Since the loss of this modification in yeast mitochondrial 21S rRNA severely affects the assembly of 54S subunits, it is likely that the analogous 2'-O-methylguanosine at position 2251 (Gm2251) in E coli 23S rRNA is also required for the assembly of 50S subunits. Gm could be a critical structural determinant for the correct folding of the rRNA, the binding of one or more ribosomal proteins, or the interaction of the rRNA with tRNA. Previous work has shown that the mitochondrial large rRNAs are minimally modified relative to the E coli and eukaryotic cytoplasmic rRNAs. By direct chemical analysis using combined high performance liquid chromatography-mass spectrometry, the modification status of the yeast mitochondrial rRNAs was reexamined, revealing the presence of Gm, Um and pseudouridine (psi) in 21S rRNA. The Um was mapped to nucleotide 2791, which corresponds to the ribose methylated and universally conserved U2552 in E coli 23S rRNA, and the psi has been recently mapped to position 2819, which corresponds to psi 2580 in E coli 23S rRNA. The retention of Um and psi nucleotides in the peptidyl transferase center of the otherwise minimally modified mitochondrial rRNAs suggests that these modifications, like Gm2270, might be essential for ribosome assembly or function or both.

Effects of amino acid side-chain volume on chain packing in genetically engineered periodic polypeptides.

The fidelity of bacterial protein synthesis allows the production of architecturally well-defined polymeric materials through precise control of chain length, sequence, stereochemistry, and interchain interactions. In the present paper, we examine the relation between amino acid residue volume and crystalline unit cell dimensions, in a set of periodic protein polymers of repeating unit sequence -(AlaGly)3-X-Gly-, where X is Asn, Phe, Ser, Val, or Tyr. The proteins were overexpressed in Escherichia coli, purified by simple procedures based on acid/ethanol precipitation or insolubility in aqueous sodium dodecyl sulfate, and processed to form oriented crystalline mats by precipitation from formic acid under mechanical shear. X-ray diffraction analyses revealed that the basic structures of the -(AlaGly)3-X-Gly- polymers are identical to that previously reported for [(AlaGly)3-GluGly]36, [Krejchi, M.T., Atkins, E.D.T., Waddon, A.J., Fournier, M.J., Mason, T.L., and Tirrell, D.A. (1994) Science 265, 1427-1432], with the oligoalanylglycine segments forming antiparallel beta-sheets and the substituted amino acids occurring within three-residue folds at the lamellar surfaces. The X-ray diffraction signals for each member of the family index on an orthorhombic unit cell; the a-axis (hydrogen bond direction) and c-axis (chain direction) spacings remain invariant but the b-axis (sheet stacking direction) spacing increases with increasing volume of the substituted amino acid. The results obtained from a variant with alternating Glu and Lys substitution at the X position, together with the results previously reported for poly(L-alanylglycine) [Panitch, A., Matsuki, K., Cantor, E.J., Cooper, S.J., Atkins, E.D.T., Fournier, M.J., Mason, T.L., and Tirrell, D.A. (1997) Macromolecules 30, 42-49] are included for comparison. The average intersheet stacking distance (b/2) increases linearly with the volume of the amino acid inserted at position X. Because the chain-folded lamellar architecture adopted by these periodic polypeptides accommodates a wide range of residues differing in charge, steric bulk, and hydrophobicity, these results illustrate a new approach to the engineering of intermolecular interactions in polymeric solids.

Chemical and biosynthetic approaches to the production of novel polypeptide materials.

Three approaches to the synthesis of the repetitive copolypeptide [(GlyAla)3-GlyProGlu]n (1) are described. Direct chemical synthesis of 1 via classical solution methods required 18 steps and afforded a polydisperse product with an average molecular weight of less than 10,000. Two alternative genetic strategies were also explored. In the first, chemically synthesized DNA oligomers were self-ligated to produce a population of multimers, which were fitted with translational start and stop signals and inserted into an expression plasmid containing the lambda PL promoter and a synthetic ribosome binding site. Transformation of E. coli led to the isolation of a stable recombinant plasmid carrying an insert encoding 12 repeats of sequence 1. Attempts to identify polypeptide 1 after induction of transformed cultures were unsuccessful. A second strategy, generating a tripartite derivative of sequence 1 carrying short N- and C-terminal extensions, afforded excellent yields of product. The relative merits of chemical and genetic approaches to repetitive polypeptide materials are discussed.

Effect of local sequence inversions on the crystalline antiparallel beta-sheet lamellar structures of periodic polypeptides: implications for chain-folding.

The crystal structure and texture of the monodisperse periodic polypeptide [(AG)3EG(GA)3EG]10 (poly(+/-AG)3EG: A=alanine, G=glycine, E=glutamic acid) were analyzed by X-ray diffraction, Fourier transform infrared spectroscopy, and electron microscopy. Structure determination was aided by comparison with the recently described structure for the related periodic polypeptide [(AG)3EG]36 by Krejchi et al. (Macromolecules 1997;30:5012). Texture-oriented samples of poly(+/-AG)3EG were obtained by crystallization of the polymer from aqueous formic acid solution. The evidence supports an antiparallel (ap) beta-sheet protein structure and the X-ray diffraction signals index on an orthorhombic unit cell with parameters: a=0.950 nm (hydrogen-bond direction), b=1.052 nm (apbeta-sheet stacking direction), c=6.95 nm (chain direction). The absence of the (010) diffraction signal, a prominent signal in the poly(AG)3EG diffraction pattern, implies that the apbeta-sheets are 'apolar', i.e. both surfaces are equally populated with alanyl methyl groups. Selective line broadening of wide-angle diffraction signals with l not equal to 0 gives an estimated crystal size of approximately/= 4 nm in the chain direction. This observation, coupled with the appearance of low-angle particle interference peaks, indicates a crystal thickness considerably less than the chain length and suggests an adjacent-re-entry chain-folded lamellar structure incorporating the apbeta-sheet architecture. The polypeptide folds through gamma-turns, in-phase with the pseudo-octapeptide repeat; the glutamic acid residues occur on the lamellar surfaces. These results and those from the crystalline lamellae of poly(AG)3EG suggest that beta-turns are not compatible with these repetitively stacked apbeta-sheet structures. This implies that intersheet interactions of alanyl methyl groups and glycyl alpha-protons are not sufficiently strong to dictate the folding geometry in these structures.

A critique of the usefulness of inferential statistics in applied behavior analysis.

Researchers continue to recommend that applied behavior analysts use inferential statistics in making decisions about effects of independent variables on dependent variables. In many other approaches to behavioral science, inferential statistics are the primary means for deciding the importance of effects. Several possible uses of inferential statistics are considered. Rather than being an objective means for making decisions about effects, as is often claimed, inferential statistics are shown to be subjective. It is argued that the use of inferential statistics adds nothing to the complex and admittedly subjective nonstatistical methods that are often employed in applied behavior analysis. Attacks on inferential statistics that are being made, perhaps with increasing frequency, by those who are not behavior analysts, are discussed. These attackers are calling for banning the use of inferential statistics in research publications and commonly recommend that behavioral scientists should switch to using statistics aimed at interval estimation or the method of confidence intervals. Interval estimation is shown to be contrary to the fundamental assumption of behavior analysis that only individuals behave. It is recommended that authors who wish to publish the results of inferential statistics be asked to justify them as a means for helping us to identify any ways in which they may be useful.

The role of stigma in reasons for HIV disclosure and non-disclosure to children.

This study examined how stigma may impact HIV-positive women's disclosure to their children. Participants included HIV-infected women recruited from AIDS service organizations located in large midwestern cities. Using a questionnaire and guided interview, quantitative data were obtained regarding perceived HIV-related stigma and rates of maternal disclosure to children aged 5-18. According to the Kruskal-Wallis test, comparison between the disclosure groups showed non-significant differences in the total score of HIV stigma (chi(2)=0.518 with df = 2, p=0.77).

Structure and function of MRP20 and MRP49, the nuclear genes for two proteins of the 54 S subunit of the yeast mitochondrial ribosome.

MRP20 and MRP49 are proteins of the large subunit of the mitochondrial ribosome in Saccharomyces cerevisiae. Their genes were identified through immunological screening of a genomic library in the expression vector lambda gt11. Nucleotide sequencing revealed that MRP49 is tightly linked to TPK3 and encodes a 16-kDa, basic protein with no significant relatedness to any other known protein. MRP20 specifies a 263-amino-acid polypeptide with sequence similarity to members of the L23 family of ribosomal proteins. The levels of the mRNAs and proteins for both MRP20 and MRP49 were regulated in response to carbon source. In [rho0] strains lacking mitochondrial rRNA, the levels of the two proteins were reduced severalfold, presumably because the unassembled proteins are unstable. Null mutants of MRP20 converted to [rho-] or [rho0], a characteristic phenotype of mutations in essential genes for mitochondrial translation. Inactivation of MRP49 caused a cold-sensitive respiration-deficient phenotype, indicating that MRP49 is not an essential ribosomal protein. The mrp49 mutants were defective in the assembly of stable 54 S ribosomal subunits at the nonpermissive temperature. With the results presented here, there are now published sequences for 14 yeast mitochondrial ribosomal proteins, only five of which bear discernable relationships to eubacterial ribosomal proteins.

Functional analysis of ribosomal protein L2 in yeast mitochondria.

The yeast nuclear gene RML2, identified through genomic sequencing of Saccharomyces cerevisiae chromosome V, was shown to encode a mitochondrial homologue of the bacterial ribosomal protein L2. Immunoblot analysis showed that the mature Rml2p is a 37-kDa polypeptide component of the mitochondrial 54 S large ribosomal subunit. Null mutants of RML2 are respiration-deficient and convert to [rho-] or [rho degrees ] cytoplasmic petites, indicating that Rml2p is essential for mitochondrial translation. RML2 is regulated transcriptionally in response to carbon source and the accumulation of Rml2p is dependent on the presence of the 21 S large rRNA. Site-directed mutagenesis showed that a highly conserved 7-amino acid sequence (Val336 to Asp342) of Rml2p is essential for function. Substitution of Gln for His-343, the most highly conserved histidine in the L2 protein family, caused cold-sensitive respiratory growth but did not affect the assembly of 54 S ribosomal subunits. Mitochondrial protein synthesis was normal in the His343 to Gln (H343Q) mutant grown at the permissive temperature (30 degrees C) and was severely impaired after growth at the nonpermissive temperature (18 degrees C). His343 corresponds to His229 in Escherichia coli L2, which has been implicated in a direct involvement in peptidyl transferase activity. The conditional phenotype of the H343Q mutant indicates that His343 is not essential for peptidyl transferase activity in yeast mitochondria.

The role of nucleotide modifications in the yeast mitochondrial ribosome.

Post-transcriptionally modified nucleotides in ribosomal RNA, and the modifying enzymes themselves, could influence the assembly, structure, and function of the ribosome. The PET56 gene in yeast encodes the enzyme responsible for formation of 2'-O-methylguanosine at a specific nucleotide in the peptidyl transferase center of the mitochondrial large subunit ribosomal RNA. While PET56 is normally essential for the formation of functional mitochondrial ribosomes, extragenic mutations have been obtained that suppress, albeit weakly, pet56 loss-of-function mutations. Thus neither the Pet56p-catalyzed ribose methylation nor the Pet56 protein itself is absolutely required for the synthesis of a functional ribosome.

Identification of the yeast nuclear gene for the mitochondrial homologue of bacterial ribosomal protein L16.

An open reading frame encoding a member of the L16 family of ribosomal proteins is adjacent to the URA7 gene on the left arm of chromosome II in Saccharomyces cerevisiae. The predicted L16-like polypeptide is basic (pl 11.12), contains 232 amino acids (26.52 kDa) and has 36% amino acid sequence identity to E. coli L16. Immunoblot analysis with polyclonal antibodies to the L16-like polypeptide showed specific cross-reaction with a 22,000 Mr mitochondrial polypeptide that co-sediments with the large subunit of the mitochondrial ribosome in sucrose density gradients. The levels of the L16 mRNA and protein varied in response to carbon source. In [rho degree] cells lacking mitochondrial rRNA, the L16 mRNA accumulated at normal levels, but the protein was barely detectable, indicating RNA-dependent accumulation of the L16 protein. Gene disruption experiments demonstrated that the yeast mitochondrial L16 is an essential ribosomal protein in vivo.

A single nucleotide substitution at the rib2 locus of the yeast mitochondrial gene for 21S rRNA confers resistance to erythromycin and cold-sensitive ribosome assembly.

We have studied a mutation (cs23) in the mitochondrial gene for 21S rRNA that affects the peptidyl transferase center of the ribosome and conditionally blocks the assembly of the 54S ribosomal subunit. Strains carrying this mutation are resistant to erythromycin and cold-sensitive for growth on nonfermentable carbon sources (Singh et al. 1978) Mitochondria isolated from mutant cells grown on glucose at 20 degrees C, the nonpermissive temperature, were depleted of the 54S subunit and instead contained a novel 45S ribosomal particle. After mutant cells were shifted from 20 degrees C to 32 degrees C, 54S subunits were assembled, apparently from the 45S particles and pre-existing ribosomal proteins. DNA sequencing revealed that the mutant phenotype is a consequence of a C to A transversion at position 3993 of the 21S rRNA gene. Previously, C to U and C to G mutations have been identified at the same position in the 21S rRNA sequence. This position corresponds to C-2611 in the E. coli 23S RNA, a nucleotide that appears to be conserved in the large rRNA of all erythromycin-sensitive ribosomes.

Accumulation of mitochondrially synthesized Saccharomyces cerevisiae Cox2p and Cox3p depends on targeting information in untranslated portions of their mRNAs.

The essential products of the yeast mitochondrial translation system are seven hydrophobic membrane proteins and Var1p, a hydrophilic protein in the small ribosomal subunit. Translation of the membrane proteins depends on nuclearly encoded, mRNA-specific translational activators that recognize the 5'-untranslated leaders of their target mRNAs. These translational activators are themselves membrane associated and could therefore tether translation to the inner membrane. In this study, we tested whether chimeric mRNAs with the untranslated sequences normally present on the mRNA encoding soluble Var1p, can direct functional expression of coding sequences specifying the integral membrane proteins Cox2p and Cox3p. DNA sequences specifying these chimeric mRNAs were inserted into mtDNA at the VAR1 locus and expressed in strains containing a nuclearly localized plasmid that supplies a functional form of Var1p, imported from the cytoplasm. Although cells expressing these chimeric mRNAs actively synthesized both membrane proteins, they were severely deficient in cytochrome c oxidase activity and in the accumulation of Cox2p and Cox3p, respectively. These data strongly support the physiological importance of interactions between membrane-bound mRNA-specific translational activators and the native 5'-untranslated leaders of the COX2 and COX3 mRNAs for localizing productive synthesis of Cox2p and Cox3p to the inner membrane.