Secondary structure features of ribosomal RNA species within intact ribosomal subunits and efficiency of RNA-protein interactions in thermoacidophilic (Caldariella acidophila, Bacillus acidocaldarius) and mesophilic (Escherichia coli) bacteria

Ribosomal subunits of Caldariella acidophila (max.growth temp., 90 degrees C) have been compared to subunits of Bacillus acidocaldarius (max. growth temp., 70 degrees C) and Escherichia coli (max. growth temp., 47 degrees C) with respect to (a) bihelical content of rRNA; (b) G . C content of bihelical domains and (c) tightness of rRNA-protein interactions. The principal results are as follows. Subunits of C. acidophilia ribosomes (Tm = 90-93 degrees C) exhibit considerable thermal tolerance over their B. acidocaldarius (Tm = 77 degrees C) and E. coli counterparts (Tm = 72 degrees C). Based on the "melting' hyperchromicities of the intact ribosomal subunits a 51-55% fraction of the nucleotides appears to participate in hydrogen-bonded base pairing regardless of ribosome source, whereas a larger fraction, 67-70%, appears to be involved in hydrogen bonding in the naked rRNA species. The G . C content of bihelical domains of both free and ribosome-bound rRNA increases with increasing thermophily; based on hyperchromicity dispersion spectra of intact subunits and free rRNA, the bihelical parts of C. acidophila rRNA are estimated to contain 63-64% G . C, compared to 58.5% G . C for B. acidocaldarius and 55% G . C for E. coli. The increment of ribosome Tm values with increasing thermophily is greater than the increase in Tm for the free rRNA, indicating that within ribosomes bihelical domains of the thermophile rRNA species are stabilized more efficiently than their mesophile counterparts by proteins or/ and other component(s). The efficiency of the rRNA-protein interactions in the mesophile and thermophile ribosomes has been probed by comparing the releases, with LiCl-urea, of the rRNA species from the corresponding ribosomal subunits stuck to a Celite column through their protein moiety; it has been established that the release of C. acidophila rRNA from the Celite-bound ribosomes occurs at salt-urea concentrations about 4-fold higher than those required to release rRNA from Celite-bound E. coli ribosomes. Compared to E. coli the C. acidophila 50 and 30 S ribosomal subunits are considerably less susceptible to treatment designed to promote ribosome unfolding through depletion of magnesium ions.     

Temperature dependent dynamics in highly homologous adenylate kinases

To correlate the structural features of enzymes to temperature adaptation, we studied psychrophile, mesophile, and thermophile adenylate kinases as model enzymes using bioinformatics and computational tools. Phylogenetic analysis revealed that mesophile and thermophile variants are clustered in one stem of phylogenetic tree and are close to contemporary time, while psychrophile enzyme is more close to their common ancestor. This finding is in good agreement with the process of environmental changes from ice age toward current warm conditions on the earth. We also performed Molecular Dynamics simulation at corresponding temperatures of all enzyme variants including 308, 318, and 328 K. It was found that mesophile enzyme has no distinct deviation of Root Mean Square Deviation (RMSD) and Radius of Gyration (Rg) values from equilibrium states at operating temperature of thermophile enzyme as well as its own optimum temperature. However, psychrophile enzyme undergoes more fluctuations with higher amplitude of change; particularly at 328 K. It was also found that initial increasing of RMSD and Rg for Psychrophile enzyme at all temperatures is occurred gradually; while, the increment of this structural parameters for thermophile enzyme at 328 K is occurred in a highly cooperative and switching manner demonstrating snap structural change of thermophile enzyme in its own temperature. By analysis of Root Mean Square Fluctuation values at different temperatures, we identified two flexible fragments in adenylate kinases so that different dynamic behavior of these regions in mesophile enzyme against operating temperatures of psychrophile and thermophile variants is critical in compensation of flexibility challenges at respective temperatures.     

Translation of Synthetic and Endogenous Messenger Ribonucleic Acid In Vitro by Ribosomes and Polyribosomes from Clostridium pasteurianum

Ribosomes and polyribosomes from Clostridium pasteurianum were isolated and their activities were compared with those of ribosomes from Escherichia coli in protein synthesis in vitro. C. pasteurianum ribosomes exhibited a high level of activity due to endogenous messenger ribonucleic acid (RNA). For translation of polyuridylic acid [poly(U)], C. pasteurianum ribosomes required a higher concentration of Mg(2+) and a much higher level of poly(U) than did E. coli ribosomes. Phage f2 RNA added to the system with C. pasteurianum ribosomes gave no significant stimulation of protein synthesis in a homologous system or with E. coli initiation factors. The 30S and 50S subunits prepared from C. pasteurianum ribosomes reassociated less readily than subunits from E. coli. The ability of the C. pasteurianum subunits to reassociated was found to be dependent upon the presence of a reducing agent during preparation and during analysis of the reassociation products. In heterologous combinations, E. coli 30S subunits associated readily with C. pasteurianum 50S subunits to form 70S particles, but C. pasteurianum 30S subunits and E. coli 50S subunits did not associate. In poly(U) translation, E. coli 30S subunits were active in combination with 50S subunits from either E. coli or C. pasteurianum, but C. pasteurianum 30S subunits were not active in combination with either type of 50S subunits. Polyribosomes prepared from C. pasteurianum were very active in protein synthesis, and well-defined ribosomal aggregates as large as heptamers could be seen on sucrose gradients. An attempt was made to demonstrate synthesis in vitro of ferredoxin.     

Secondary structure features of ribosomal RNA species within intact ribosomal subunits and efficiency of RNA-protein interactions in thermoacidophilic (Caldariella acidophila,Bacillus acidocaldarius) and mesophilic (Escherichia coli) bacteria

Ribosomal subunits of Caldariella acidophila (max.growth temp., 90°C) have been compared to subunits of Bacillus acidocaldarius (max. growth temp., 70°C) and Escherichia coli (max. growth temp., 47°C) with respect to (a) bihelical content of rRNA; (b) G·C content of bihelical domains and (c) tightness of rRNA-protein interactions. The principal results are as follows. 1. Subunits of C. acidophila ribosomes (T_m = 90–93°C) exhibit considerable thermal tolerance over their B. acidocaldarius (T_m = 77°C) and E. coli counterparts (T_m = 72°C). 2. Based on the ‘melting’ hyperchromicities of the intact ribosomal subunits a 51–55% fraction of the nucleotides appears to participate in hydrogen-bonded base pairing regardless of ribosome source, whereas a larger fraction, 67–70%, appears to be involved in hydrogen bonding in the naked rRNA species. 3. The G·C content of bihelical domains of both free and ribosome-bound rRNA increases with increasing thermophily; based on hyperchromicity dispersion spectra of intact subunits and free rRNA, the bihelical parts of C. acidophila rRNA are estimated to contain 63–64% G·C, compared to 58.5% G·C for B. acidocaldarius and 55% G·C for E. coli. 4. The increment in ribosome T_m values with increasing thermophily is greater than the increase in T_m for the free rRNA, indicating that within ribosomes bihelical domains of the thermophile rRNA species are stabilized more efficiently than their mesophile counterparts by proteins or/ and other component(s). 5. The efficiency of the rRNA-protein interactions in the mesophile and thermophile ribosomes has been probed by comparing the releases, with LiCl-urea, of the rRNA species from the corresponding ribosomal subunits stuck to a Celite column through their protein moiety; it has been established that the release of C. acidophila rRNA from the Celite-bound ribosomes occurs at salt-urea concentrations about 4-fold higher than those required to release rRNA from Celite-bound E. coli ribosomes. 6. Compared to E. coli, the C. acidophila 50 and 30 S ribosomal subunits are considerably less susceptible to treatment designed to promote ribosome unfolding through depletion of magnesium ions.     

Comparison of ribosomes from Coxiella burnetii and Escherichia coli by gel electrophoresis, protein synthesis, and immunological techniques.

Ribosomes and postribiosomal supernatant fluid (S-100) were isolated from Coxiella burnetii. The ribosomes functioned in polyuridylic acid-directed polyphenylalanine synthesis in the presence of S-100 from either C. burnetii or Escherichia coli. C. burnetii S-100 promoted translation with E. coli ribosomes. Antisera against E. coli elongation factor G and ribosomal proteins L7/L12 cross-reacted with rickettsial S-100 and ribosomes, respectively. Ribosomal proteins were analyzed by two-dimensional gel electrophoresis.     

A totally synthetic histidine-2 ferredoxin: thermal stability and redox properties.

The entire polypeptide of Clostridium pasteurianum ferredoxin (Fd) with a site-substituted tyrosine-2----histidine-2 was synthesized using standard t-Boc procedures, reconstituted to the 2[4Fe-4S] holoprotein, and compared to synthetic C. pasteurianum and native Fds. Although histidine-2 is commonly found in thermostable clostridial Fds, the histidine-2 substitution into synthetic C. pasteurianum Fd did not significantly increase its thermostability. The reduction potential of synthetic histidine-2 Fd was -343 and -394 mV at pH 6.4 and 8.7, respectively, versus standard hydrogen electrode. Similarly, Clostridium thermosaccharolyticum Fd which naturally contains histidine-2 was previously determined to have a pH-dependent reduction potential [Smith, E.T., & Feinberg, B.A. (1990) J. Biol. Chem. 265, 14371-14376]. An electrostatic model was used to calculate the observed change in reduction potential with pH for a homologous ferredoxin with a known X-ray crystal structure containing a hypothetical histidine-2. In contrast, the reduction potential of both native C. pasteurianum Fd and synthetic Fd with the C. pasteurianum sequence was -400 mV versus standard hydrogen electrode and was pH-independent [Smith, E.T., Feinberg, B.A., Richards, J.H., & Tomich, J.M. (1991) J. Am. Chem. Soc. 113, 688-689]. On the basis of the above results, we conclude that the observed pH-dependent reduction potential for both synthetic and native ferredoxins that contain histidine-2 is attributable to the electrostatic interaction between histidine-2 and iron-sulfur cluster II which is approximately 6 A away.     

Comparison of the stability of phycocyanins from thermophilic, mesophilic, psychrophilic and halophilic algae.

Protein unfolding of eight different phycocyanins was investigated utilizing circular dichroism and visible spectra. The phycocyanin samples were extracted from algae that are normally found in vastly different environments, and are classified as mesophilic, thermophilic, halophilic and psychrophilic. The ability of these proteins to resist the denaturant urea is in the order of thermophile greater than mesophile, halophile greater than psychrophile. Based on a two-state approximation the apparent free energies of protein unfolding at zero urea denaturant concentration, deltaGH2Oapp, were found to range from 2.4 to 8.8 kcal/mole for the eight phycocyanins at pH 6 and 25 degrees C. The proteins from the thermophile are generally more stable than those from the mesophile. An extra stability of the halophile is believed due to the specific interaction of the proteins and the ions in solution. A correction for deltaGH2Oapp due to minor amino acid differences reveals that the stability and the structural properties of these proteins are primarily affected by this minor difference in amino acid compositions.     

The universal ancestor was a thermophile or a hyperthermophile: tests and further evidence

The existence of a correlation between the optimal growth temperature of various organisms and a thermophily index (based on the propensity of amino acids to enter more frequently into the proteins of thermophiles/hyperthermophiles) allows inferences to be made on the mesophilic or thermophilic nature of the last universal common ancestor (LUCA). By reconstructing the ancestral sequences of the various ancestors using methods based on maximum likelihood and maximum parsimony, these sequences can be attributed to the mesophiles or (hyper)thermophiles and the following conclusions can be drawn. (1) There is no evidence that the LUCA might have been a mesophile and observations seem to imply that the LUCA was a thermophile or a hyperthermophile; (2) The ancestors of the Archaea and Bacteria domains seem to be (hyper)thermophiles while that of the Eukarya domain turns out to be a mesophile. These conclusions are independent of both (i) where the root is located on the topology of the universal tree (based on that of the small subunit ribosomal RNA) and (ii) the presence of hyperthermophile bacteria near the node of the Bacteria domain ancestor. These conclusions are easier to interpret in the light of the hypotheses that see the origin of life taking place at a high temperature.     

Stability of ribosomes and ribosomal ribonucleic acid from Bacillus stearothermophilus

After heating at 65 C, ribosomes isolated from Bacillus stearothermophilus were strikingly more heat-stable than comparable preparations from Escherichia coli when tested for ability to support polyuridylic acid-directed phenylalanine incorporation at 37 C. The stability of ribosomes was also determined by measurements of hyperchromicity at 259 mmu while heating them from 25 to 90 C. In standard buffer containing 0.01 m Mg(++), the T(m) (temperature at the midpoint of total hyperchromicity) of E. coli and B. stearothermophilus ribosomes was 71 and 81 C, respectively. In a magnesium-free buffer, the T(m) of E. coli and B. stearothermophilus ribosomes was 44 and 64 C, respectively. Putrescine (0.01 m) was more effective in stabilizing ribosomes from B. stearothermophilus than those from E. coli. Spermidine (0.001 m), on the other hand, was more effective in stabilizing ribosomes from E. coli than those from B. stearothermophilus. Melting curves of total ribosomal ribonucleic acid (rRNA) from E. coli and B. stearothermophilus revealed T(m) values of 50 and 60 C, respectively. Putrescine stabilized thermophile rRNA, but had no effect on E. coli rRNA. Sucrose density gradients demonstrated that thermophile 23S ribonucleic acid was degraded during storage at -20 C; the 23S component from E. coli was stable under these conditions. The results are discussed in terms of the mechanism of ribosome heat stability and the role of the ribosome in governing the temperature limits for bacterial growth.     

Fatty Acid Composition of Thermophilic, Mesophilic, and Psychrophilic Clostridia

The fatty acid composition of two thermophilic anaerobes was determined, and the results were compared with those from a mesophilic and a psychrophilic anaerobe. Notable differences were that the thermophiles contained a higher content of saturated straight- and branched-chain fatty acids, and, of the latter, iso C₁₅ was the predominant type. The mesophile and psychrophile were characterized by having a higher percentage of unsaturated fatty acids. An unidentified fatty acid, present in all of the organisms, was purified from the psychrophile. By physical and chemical analysis the structure of the unknown acid was resolved and found to be the unsaturated cyclopropane fatty acid, 12,13-methylene-9-tetradecenoic acid.     

Occurrence and molecular characterization of cultivable mesophilic and thermophilic obligate anaerobic bacteria isolated from paper mills

The aim of this work was to characterize the cultivable obligate anaerobic bacterial population in paper mill environments. A total of 177 anaerobically grown bacterial isolates were screened for aerotolerance, from which 67 obligate anaerobes were characterized by automated ribotyping and 41 were further identified by partial 16S rDNA sequencing. The mesophilic isolates indicated 11 different taxa (species) within the genus Clostridium and the thermophilic isolates four taxa within the genus Thermoanaerobacterium and one within Thermoanaerobacter (both formerly Clostridium). The most widespread mesophilic bacterium was closely related to C. magnum and occurred in three of four mills. One mill was contaminated with a novel mesophilic bacterium most closely related to C. thiosulfatireducens. The most common thermophile was T. thermosaccharolyticum, occurring in all four mills. The genetic relationships of the mill isolates to described species indicated that most of them are potential members of new species. On the basis of identical ribotypes clay could be identified to be the contamination source of thermophilic bacteria. Automated ribotyping can be a useful tool for the identification of clostridia as soon as comprehensive identification libraries are available.     

Archaeal RecA homologues: different response to DNA-damaging agents in mesophilic and thermophilic Archaea

Two archaeal proteins, RadA and RadB, share similarity with the RecA/Rad51 family of recombinases, with RadA being the functional homologue. We have studied and compared the RadA and RadB proteins of mesophilic and thermophilic Archaea. In growing cells, RadA levels are similar in mesophilic Methanococcus species and the hyperthermophile Methanococcus jannaschii. Treatment of cells with mutagenic agents (methylmethane sulfonate or UV light) increased the expression of RadA (as evidenced by higher levels of both mRNA and protein) in all organisms tested, but the increase was greater in the mesophiles than in the thermophiles M. jannaschii and Sulfolobus solfataricus. Recombinantly expressed RadA proteins from the mesophile M. voltae and the thermophile M. jannaschii were similar in their ATPase- and DNA-binding activities. All the data are consistent with proposals that RadA plays the same role as eukaryotic Rad51. Surprisingly, the data also suggested that the thermophiles do not need more RadA protein or activity than the mesophiles. On the other hand, RadB is not coregulated with RadA, and its role remains unclear. Neither RadA nor RadB from a mesophile or from a thermophile rescued the UV-sensitive phenotype of an Escherichia coli recA- host.     

Substitution of aspartic acid with glutamic acid increases the unfolding transition temperature of a protein

Proteins from thermophiles are more stable than those from mesophiles. Several factors have been suggested as causes for this greater stability, but no general rule has been found. The amino acid composition of thermophile proteins indicates that the content of polar amino acids such as Asn, Gln, Ser, and Thr is lower, and that of charged amino acids such as Arg, Glu, and Lys is higher than in mesophile proteins. Among charged amino acids, however, the content of Asp is even lower in thermophile proteins than in mesophile proteins. To investigate the reasons for the lower occurrence of Asp compared to Glu in thermophile proteins, Glu was substituted with Asp in a hyperthermophile protein, MjTRX, and Asp was substituted with Glu in a mesophile protein, ETRX. Each substitution of Glu with Asp decreased the Tm of MjTRX by about 2 degrees C, while each substitution of Asp with Glu increased the Tm of ETRX by about 1.5 degrees C. The change of Tm destabilizes the MjTRX by 0.55 kcal/mol and stabilizes the ETRX by 0.45 kcal/mol in free energy.     

Structure and composition of the photochemical apparatus of the antarctic green alga,Chlamydomonas subcaudata

The green alga, Chlamydomonas subcaudata, collected from a perennially ice-covered Antarctic lake, was able to grow at temperatures of 16°C or lower, but not at temperatures of 20°C or higher, which confirmed its psychrophilic nature. Low temperature (77 K) Chl a fluorescence emission spectra of whole cells of the mesophile, C. reinhardtii, indicated the presence of major emission bands at 681 and 709 nm associated with PS II and PS I, respectively. In contrast, emission spectra of whole cells of C. subcaudata exhibited major emission bands at 681 and 692 nm associated with PS II, but the absence of a major PS I emission band at 709 nm. These results for C. subcaudata were consistent with: (1) low ratio of Chl a/b (1.80); (2) low levels of PsaA/PsaB heterodimer as well as specific Lhca polypeptides as determined by immunoblotting, (3) decreased levels of the Chl-protein complexes CP1 and LHC I associated with PS I; and (4) an increased stability of the oligomeric form of LHC II as assessed by non-denaturing gel electrophoresis in the psychrophile compared to the mesophile. Furthermore, immunoblotting indicated that the stoichiometry of PS II:PS I:CF₁ is significantly altered in C. subcaudata compared to the mesophile. Even though the psychrophile is adapted to growth at low irradiance, it retained the capacity to adjust the total xanthophyll cycle pool size as well as the epoxidation state of the xanthophyll cycle. Despite these differences, the psychrophile and mesophile exhibited comparable photosynthetic efficiency for O₂ evolution regardless of growth conditions. P^max for both Chlamydomonas species was similar only when grown under identical conditions. We suggest that these photosynthetic characteristics of the Antarctic psychrophile reflect the unusual light and low temperature regime to which it is adapted.     

Crowding in extremophiles: linkage between solvation and weak protein-protein interactions, stability and dynamics, provides insight into molecular adaptation

The study of the molecular adaptation of microorganisms to extreme environments (solvent, temperature, etc.) has provided tools to investigate the complex relationships between protein-solvent and protein-protein interactions, protein stability and protein dynamics, and how they are modulated by the crowded environment of the cell. We have evaluated protein-solvent and protein-protein interactions by solution experiments (analytical ultracentrifugation, small angle neutron and X-ray scattering, density) and crystallography, and protein dynamics by energy resolved neutron scattering. This review concerns work from our laboratory on (i) proteins from extreme halophilic Archaea, and (ii) psychrophile, mesophile, thermophile and hyperthermophile bacterial cells.     

Characterization and Stability of Ribosomes from Mesophilic and Thermophilic Bacteria

Ribosomes were isolated from three mesophilic and three thermophilic strains of Bacillus. The ribosomes consisted of about 55% protein and 45% ribonucleic acid. Average ratios for the absorbance at 260/235 and 260/280 mmu were 1.77 and 1.92 for the mesophiles and 1.63 and 1.84 for the thermophiles. Ultracentrifugation revealed mainly components with sedimentation coefficients of about 30, 50, 70, 100, and 120S. All the preparations were shown to contain a ribonuclease which, in the presence of ethylenediaminetetraacetic acid, led to ribosome breakdown as measured by the increase in acid-soluble nucleotides. The stability of the ribosomes from the thermophiles was consistently greater than that of the ribosomes from the mesophiles. After 5 hr at 37 C, the breakdown was about 80% for the ribosomes from the mesophiles and 55 to 70% for those from the thermophiles. At 60 C, the ribosomes from the mesophiles were broken down slightly more and at a faster rate than those from the thermophiles. At temperatures above 60 C, the breakdown was again more pronounced for the ribosomes from the mesophiles.     

Redox properties of several bacterial ferredoxins using square wave voltammetry

The equilibrium reduction potential of the 2[4Fe-4S] ferredoxin (Fd) isolated from four different bacterial strains was determined at a methyl viologen-modified gold electrode using square wave voltammetry. The observed reduction potential at pH 8 for Clostridium thermoaceticum Fd was -385 mV; Clostridium pasteurianum, -393 mV; Clostridium thermosaccharolyticum, -408 mV; and Chromatium vinosum, -460 mV versus normal hydrogen electrode at 25 degrees C. The reduction potential of the C. pasteurianum Fd was found to be pH independent from pH 6.4 to 8.7, indicating that the electron transfer mechanism does not involve proton exchange. In contrast, the reduction potential of the C. thermosaccharolyticum Fd was found to be pH dependent from pH 6.4 to 8.7, with pKox approximately 7 and pKred approximately 7.5. The +30 mV change in reduction potential from pH 8.7 to 6.4 was attributed to an electrostatic interaction between the iron-sulfur cluster II and the protonated histidine 2 residue located about 6 A away. The Ch. vinosum Fd interacted reversibly at the methyl viologen-modified gold electrode, and its reduction potential was verified using visible spectroelectrochemistry. The reduction potential of Ch. vinosum Fd was found to be 30 mV more positive than previously reported. The similarities of the bacterial Fd reduction potentials are discussed in terms of the homology of their primary structure as reflected by the similarities in the visible and circular dichroic spectra.     

Site of initial glycosylation of mannoproteins from Saccharomyces cerevisiae.

The cellular site of initial glycosylation of proteins from Saccharomyces cerevisiae has been studied. Short pulses of [U-14C]mannose label the ribosomal fraction of the yeast. Most of the label was associated with polysomes; monosomes contained only a small amount of radioactivity. All of the radioactivity present in the polysomal fraction was accounted by mannose and smaller amounts of glucose and glucosamine. Puromycin treatment detached more than 50% of the radioactivity from the polysomes; treatment of polysomes at pH 10.0 also caused the release of radioactivity. These results indicate that initial sugar binding occurs while the nascent polypeptide chains are still growing on the ribosomes. When the cells were preincubated with 2-deoxy-D-glucose, incorporation of [U-14C]mannose into the polysomes and the cell wall was inhibited, whereas its incorporation into membrane fractions was unimpaired. It was concluded that 2-deoxy-D-glucose inhibited the synthesis of glycoproteins by interference with the initial glycosylation steps at the ribosomal level.     

Two cellulolytic Clostridium species: Clostridium cellulosi sp. nov. and Clostridium cellulofermentans sp. nov.

Two cellulolytic clostridia, one thermophilic and the other mesophilic, were isolated and characterized. Cells of the thermophile are gram-negative rods that are motile with lophotrichous flagella and spherical terminal endospores which swell the cells. The optimum growth temperature is 55 to 60 degrees C, with a range of 40 to 65 degrees C. The deoxyribonucleic acid composition is 35 mol% G + C. The name Clostridium cellulosi sp. nov. is proposed. The type strain is AS 1.1777. Cells of the mesophile are gram negative and motile with peritrichous flagella and terminal oval or spherical spores which swell the cells. The deoxyribonucleic acid composition is 34 mol% G + C. The name Clostridium cellulofermentans sp. nov. is proposed. The type strain is AS 1.1775. Both C. cellulosi AS 1.1777 and C. cellulofermentans AS 1.1775 are deposited in the China Committee for Culture Collection of Microorganisms, Institute of Microbiology, Academia Sinica, Beijing, People's Republic of China.     

Properties of cell-free extracts fromBacillus subtilis and its thermophilic mutant

After being heated at 65°C for 10 min, 51% of the protein in a cell-free extract fromBacillus subtilis BR151 was denatured, whereas the comparable value was 8% for the S-30 of a spontaneously occurring, temperature-resistant (T/r) mutant. Although ribosomes isolated from the T/r mutant retained 97% of their initial protein synthetic activity when preincubated at 60°C for 30 min, ribosomes prepared from the mesophilic parent were completely inactivated under these conditions. The optimum temperature for poly U-directed phenylalanine incorporation was 45°C for both parental and mutant extracts assayed in the absence of polyamines. The addition of spermidine to the S-30 from the mesophilic parent inhibited protein synthesis at each temperature tested, whereas this polyamine stimulated polyphenylalanine synthesis in the T/r extract at both 55°C and 65°C.