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A system for the analysis of yeast ribosomal DNA mutations.

To develop a system for the analysis of eucaryotic ribosomal DNA (rDNA) mutations, we cloned a complete, transcriptionally active rDNA unit from the yeast Saccharomyces cerevisiae on a centromere-containing yeast plasmid. To distinguish the plasmid-derived ribosomal transcripts from those encoded by the rDNA locus, we inserted a tag of 18 base pairs within the first expansion segment of domain I of the 26S rRNA gene. We demonstrate that this insertion behaves as a neutral mutation since tagged 26S rRNA is normally processed and assembled into functional ribosomal subunits. This system allows us to study the effect of subsequent mutations within the tagged rDNA unit on the biosynthesis and function of the rRNA. As a first application, we wanted to ascertain whether the assembly of a 60S subunit is dependent on the presence in cis of an intact 17S rRNA gene. We found that a deletion of two-thirds of the 17S rRNA gene has no effect on the accumulation of active 60S subunits derived from the same operon. On the other hand, deletions within the second domain of the 26S rRNA gene completely abolished the accumulation of mature 26S rRNA.

Base Sequence↗

On molecular taxonomy: what is in a name?

Gene sequences of small portions of the genome are often used for premature detailed taxonomic changes, neglecting polyphasic taxonomy, which should also consider phenotypical characteristics. Three examples are given: (i) Recently, members of the genera Eperythrozoon and Haemobartonella have been moved, correctly so, from the Rickettsiales to the Mycoplasmatales, but were assigned to the genus Mycoplasma, mostly on the basis of 16S rRNA sequence analysis. Not only is the 16S rRNA sequence similarity between 'classical' Mycoplasma and these species of Eperythrozoon and Haemobartonella less than that between some other well-recognised bacterial genera, but their biological differences amply justify their classification in different genera of the Mycoplasmatales. Furthermore, the move creates considerable confusion, as it necessitates new names for some species, with more confusion likely to come when the 16S rRNA sequences of the type species of Eperythrozoon, a name which has priority over Mycoplasma, will be analysed. (ii) In the Rickettsiales, members of the genera Anaplasma, Ehrlichia, Cowdria, Neorickettsia and Wolhbachia are so closely related phylogenetically on the basis of 16S rRNA sequences, and for some also of groESL operon sequences, that they have recently been fused, correctly so, into one family, the Anaplasmataceae, while the tribes Ehrlichieae and Wolbachieae have been abolished. Sequence diversity within the 'classical' genus Ehrlichia has led to classifying E. phagocytophila (including E. equi and the agent of human granulocytic ehrlichiosis), E. platys and E. bovis in the genus Anaplasma, while others have been retained in Ehrlichia, which also includes Cowdria ruminantium. E. sennetsu and E. risticii have been transferred to the genus Neorickettsia. 16S rRNA and GroEL sequences of 'classical' Anaplasma and some members of 'classical' Ehrlichia do show a close relationship, but differences in citrate synthase gene sequences, the GC content of this gene, and sequences of the gene encoding the beta-subunit of RNA polymerase, not to speak of the phenotypical differences, do not justify the fusion into one genus. Because of the phylogenetical diversity in Ehrlichia it is recommended that a new genus name be created for the E. phagocytophila genogroup (and E. platys and E. bovis). (iii) One of the conclusions of studies on the phylogeny of ticks of the subfamilies Rhipicephalinae and Hyalomminae, based on nucleotide sequences from 12S rRNA, cytochrome c oxidase I, the internal transcribed spacer 2, 18S rRNA, as well as morphological characters, is that Boophilus should be considered as a subgenus of Rhipicephalus. While Boophilus and Rhipicephalus are undoubtedly close, the obviously important morphological and biological differences between the genera Rhipicephalus and Boophilus are thus overruled by similarities in the sequences of a number of genes and this leads to considerable confusion. Polyphasic taxonomy amply justifies maintaining Boophilus as a separate genus, phylogenetically near to Rhipicephalus. This note is a plea for a cautious and balanced approach to taxonomy, taking into account molecular genotypical information, as far as is possible from different genes, as well as phenotypical characteristics.

Anaplasmataceae↗

Variation in 16S-23S rRNA intergenic spacer regions in Photobacterium damselae: a mosaic-like structure.

Phenotypically, Photobacterium damselae subsp. piscicida and P. damselae subsp. damselae are easily distinguished. However, their 16S rRNA gene sequences are identical, and attempts to discriminate these two subspecies by molecular tools are hampered by their high level of DNA-DNA similarity. The 16S-23S rRNA internal transcribed spacers (ITS) were sequenced in two strains of Photobacterium damselae subsp. piscicida and two strains of P. damselae subsp. damselae to determine the level of molecular diversity in this DNA region. A total of 17 different ITS variants, ranging from 803 to 296 bp were found, some of which were subspecies or strain specific. The largest ITS contained four tRNA genes (tDNAs) coding for tRNA(Glu(UUC)), tRNA(Lys(UUU)), tRNA(Val(UAC)), and tRNA(Ala(GGC)). Five amplicons contained tRNA(Glu(UUC)) combined with two additional tRNA genes, including tRNA(Lys(UUU)), tRNA(Val(UAC)), or tRNA(Ala(UGC)). Five amplicons contained tRNA(Ile(GAU)) and tRNA(Ala(UGC)). Two amplicons contained tRNA(Glu(UUC)) and tRNA(Ala(UGC)). Two different isoacceptor tRNA(Ala) genes (GGC and UGC anticodons) were found. The five smallest amplicons contained no tRNA genes. The tRNA-gene combinations tRNA(Glu(UUC))-tRNA(Val(UAC))-tRNA(Ala(UGC)) and tRNA(Glu(UUC))-tRNA(Ala(UGC)) have not been previously reported in bacterial ITS regions. The number of copies of the ribosomal operon (rrn) in the P. damselae chromosome ranged from at least 9 to 12. For ITS variants coexisting in two strains of different subspecies or in strains of the same subspecies, nucleotide substitution percentages ranged from 0 to 2%. The main source of variation between ITS variants was due to different combinations of DNA sequence blocks, constituting a mosaic-like structure.

Base Sequence↗

Structure and organization of a cluster of sic tRNA genes in the space between tandem ribosomal RNA gene sets in Bacillus subtilis.

Hybridization of Southern blots of EcoRI digests of total Bacillus subtilis DNA with ribosomal RNA and transfer RNA probes provides evidence of highly clustered tRNA and rRNA genes, with several tRNA clusters being located in spaces which span between tandem ribosomal RNA gene sets. Clones containing these tRNA clusters were isolated from a Charon 4A library. One of them, denoted trrnB, was partially sequenced. A cluster of six tRNA genes was found, with anticodon assignments of Asn, Thr, Gly, Arg, Pro, Ala. This cluster is closely flanked on both sides by ribosomal RNA gene sets, which were identified by sequencing upward through the 5 S rRNA gene, across a space, and into the 23 S rRNA gene, and also sequencing downward into the 16 S rRNA gene. The tRNA gene cluster appears to be organized into at least two transcriptional units separated by an attenuator region. These transcriptional units may be components of the flanking ribosomal RNA operons. The putative promoter region of the downstream 16 S rRNA is organized differently from Escherichia coli; it is smaller and seems less complex. This gene organization provides insight into possible mechanisms for coordinate and differential control of transfer and ribosomal RNA gene expression.

Anticodon↗

Sequence variation of the 16S to 23S rRNA spacer region in Salmonella enterica.

The possibility for identification of Salmonella enterica serotypes by sequence analysis of the 16S to 23S rRNA internal transcribed spacer was investigated by direct sequencing of polymerase chain reaction-amplified DNA from all operons simultaneously in a collection of 25 strains of 18 different serotypes of S. enterica, and by sequencing individual cloned operons from a single strain. It was only possible to determine the first 117 bases upstream from the 23S rRNA gene by direct sequencing because of variation between the rrn operons. Comparison of sequences from this region allowed separation of only 15 out of the 18 serotypes investigated and was not specific even at the subspecies level of S. enterica. To determine the differences between internal transcribed spacers in more detail, the individual rrn operons of strain JEO 197, serotype IV 43:z4,z23:-, were cloned and sequenced. The strain contained four short internal transcribed spacer fragments of 382-384 bases in length, which were 98.4-99.7% similar to each other and three long fragments of 505 bases with 98.0-99.8% similarity. The study demonstrated a higher degree of interbacterial variation than intrabacterial variation between operons for serotypes of S. enterica.

Base Sequence↗

The trmA promoter has regulatory features and sequence elements in common with the rRNA P1 promoter family of Escherichia coli.

The tRNA(m5U54)methyltransferase, whose structural gene is designated trmA, catalyzes the formation of 5-methyluridine in position 54 of all tRNA species in Escherichia coli. The synthesis of this enzyme has previously been shown to be both growth rate dependent and stringently regulated, suggesting regulatory features similar to those of rRNA. We have determined the complete nucleotide sequence of the trmA operon in E. coli and the sequence of the trmA promoter region in Salmonella typhimurium and also analyzed the transcriptional regulation of the gene. The trmA and the btuB (encoding the vitamin B12 outer membrane receptor protein) promoters are divergent promoters separated by 102 bp between the transcriptional start sites. The trmA promoters of both E. coli and S. typhimurium share promoter elements with the rRNA P1 promoter. The sequence downstream from the -10 region of the trmA promoter is homologous to the discriminatory region found in stringently regulated promoters. Next to and upstream from the -10 region is a sequence, TCCC, in the trmA promoter that is present in all of the seven rRNA P1 promoters and in some tRNA promoters but not in any other sigma 70 promoter. However, a similar motif is also found in promoters transcribed by the heat shock sigma factor sigma 32. The trmA gene is transcribed as a monocistronic operon, and the 3' end of the transcript is shown to be located downstream from a dyad symmetry region not followed by a poly(U) stretch. Using a trmA-cat operon fusion, we show that the growth rate-dependent regulation of trmA resembles that of rRNA and operates at the level of transcription.

Amino Acid Sequence↗

Evidence for unlinked rrn operons in the Planctomycete Pirellula marina.

Southern hybridization of rRNAs to chromosomal BamHI-digested DNA of the eubacterium Pirellula marina revealed the presence of two sets of 16S and 23S rRNA genes. The two copies of the 23S rRNA genes, located on 11- and about 13-kilobase (kb) inserts, were isolated from a lambda bacteriophage Charon 35 library. The 11-kb fragment was cloned directly into pBR322, while a 5.4-kb BamHI-PstI rDNA subfragment of the approximately 13-kb insert was cloned into pUC18. Both recombinant plasmids, pPI1100 and pPI540, were characterized by restriction enzyme mapping and Southern hybridization with the large rRNA species. Restriction fragments from both inserts were subcloned into phage M13 mp18 and mp19. Correlation of genomic hybridization data with physical characterization of recombinant plasmids showed that, in contrast to the general organization of rrn operons in eubacteria, the 16S rRNA genes of P. marina are separated by at least 8.5 (pPI540) and 4.4 (pPI1100) kb, respectively, from the closely linked 23S-5S rRNA genes. Comparison of the flanking regions from both 23S-5S rRNA genes with published consensus sequences of structural elements indicates the presence of putative transcription signals, i.e., a single Pribnow box, discriminator, antitermination boxes A, B, and C, and a Rho-independent terminator.

Base Sequence↗

The solution structure of ribosomal protein S4 delta41 reveals two subdomains and a positively charged surface that may interact with RNA.

S4 is one of the first proteins to bind to 16S RNA during assembly of the prokaryotic ribosome. Residues 43-200 of S4 from Bacillus stearothermophilus (S4 Delta41) bind specifically to both 16S rRNA and to a pseudoknot within the alpha operon mRNA. As a first step toward understanding how S4 recognizes and organizes RNA, we have solved the structure of S4 Delta41 in solution by multidimensional heteronuclear nuclear magnetic resonance spectroscopy. The fold consists of two globular subdomains, one comprised of four helices and the other comprised of a five-stranded antiparallel beta-sheet and three helices. Although cross-linking studies suggest that residues between helices alpha2 and alpha3 are close to RNA, the concentration of positive charge along the crevice between the two subdomains suggests that this could be an RNA-binding site. In contrast to the L11 RNA-binding domain studied previously, S4 Delta41 shows no fast local motions, suggesting that it has less capacity for refolding to fit RNA. The independently determined crystal structure of S4 Delta41 shows similar features, although there is small rotation of the subdomains compared with the solution structure. The relative orientation of the subdomains in solution will be verified with further study.

Amino Acid Sequence↗

Phylogenetic comparison of the cyanobacterial genera Anabaena and Aphanizomenon.

Morphological analysis and sequencing of the 165 rRNA gene, the spacer region of the ribosomal operon (ITS1) and the rbcLX (RubisCO) region was performed on 26 Anabaena strains and 14 Aphanizomenon strains isolated from several lakes in Denmark, Finland and France. Based on their morphology, Anabaena strains differed from strains of Aphanizomenon: the vegetative cells, heterocysts and akinetes were significantly wider in Anabaena than in Aphanizomenon. Phylogenetic trees based on the 16S rDNA, ITS1 and rbcLX regions showed that the planktic Anabaena strains were not distinguishable from Aphanizomenon strains. The results of the clustering of Anabaena and Aphanizomenon strains based on 16S rDNA sequences showed that these two genera are not monophyletic. Sequence analysis of the 16S rDNA, ITS1-S and rbcLX regions of the planktic Anabaena strains showed that this genus is heterogeneous. In all methods, Anabaena strains that produced different toxic compounds (e.g. anatoxin-a, microcystin and an unknown neurotoxin) were clustered separately from each other but were grouped either with non-toxic Anabaena and/or Aphanizomenon strains. Our data suggest that the planktic Anabaena and Aphanizomenon isolates belong to the same genus, regardless of their morphological differences. Thus, a taxonomic revision of the two genera is required.

Anabaena↗

Delineation of the genus Actinobacillus by comparison of partial infB sequences.

A 426 bp fragment of infB, a housekeeping gene that encodes translation initiation factor 2, was sequenced from 59 clinical isolates and type strains of Actinobacillus species and sequences were compared. Partial sequences of 16S rRNA genes were also obtained. By comparing infB sequences, Actinobacillus pleuropneumoniae, Actinobacillus equuli, Actinobacillus suis, Actinobacillus ureae, Actinobacillus arthritidis, Actinobacillus hominis and two unnamed genomospecies showed more than 85 % similarity to the type strain of the type species of the genus, Actinobacillus lignieresii. The taxonomic position of Actinobacillus capsulatus was unresolved; this species is more remotely related to A. lignieresii. The two species A. lignieresii and A. pleuropneumoniae could not be clearly separated by infB sequence analysis. The phylogeny of the genus Actinobacillus based on infB analysis was essentially congruent with relationships inferred from 16S rRNA sequence comparisons and DNA hybridization studies. Discrepancies were encountered with single strains or taxa at the periphery of the genus. Greater intraspecies variation was observed with infB sequences than with 16S rRNA gene sequences, with notable exceptions. The apparent subdivision of some species by 16S rRNA analysis was most likely caused by RNA operon heterogeneity.

Actinobacillus↗

Identification of cold shock gene loci in Sinorhizobium meliloti by using a luxAB reporter transposon.

Using a luxAB reporter transposon, seven mutants of Sinorhizobium meliloti were identified as containing insertions in four cold shock loci. LuxAB activity was strongly induced (25- to 160-fold) after a temperature shift from 30 to 15 degrees C. The transposon and flanking host DNA from each mutant was cloned, and the nucleic acid sequence of the insertion site was determined. Unexpectedly, five of the seven luxAB mutants contained transposon insertions in the 16S and 23S rRNA genes of two of the three rrn operons of S. meliloti. Directed insertion of luxAB genes into each of the three rrn operons revealed that all three operons were similarly affected by cold shock. Two other insertions were found to be located downstream of a homolog of the major Escherichia coli cold shock gene, cspA. Although the cold shock loci were highly induced in response to a shift to low temperature, none of the insertions resulted in a statistically significant decrease in growth rate at 15 degrees C.

Bacterial Proteins↗

Isolation and characterization of a new strain of Ehrlichia chaffeensis from a patient with nearly fatal monocytic ehrlichiosis.

Ehrlichia chaffeensis is the causative agent of human monocytic ehrlichiosis, a disease that ranges in severity from asymptomatic infection to death. Only one isolate of E. chaffeensis has been made, the Arkansas strain, upon which all characterizations of the agent of human monocytic ehrlichiosis have been based. We report the isolation and characterization of a new strain of E. chaffeensis, the 91HE17 strain, which was cultivated from a patient with a nearly fatal illness. The new isolate grows best in culture with careful control of pH. The two isolates are nearly identical as determined by light and electron microscopy and have significant antigenic identity in fluorescent-antibody and immunoblot assays using polyclonal antisera and the E. chaffeensis-specific monoclonal antibody 1A9. Isolate 91HE17 had 99.9% nucleotide sequence identity with the Arkansas strain in the 16S rRNA gene. Parts of the Escherichia coli GroE operon homologs had identical restriction enzyme digestion patterns, and a 425-bp region of the GroEL gene had at least 99.8% sequence identity between the E. chaffeensis Arkansas and 91HE17 strains. Isolate 91HE17 lacked an epitope identified in E. chaffeensis Arkansas by the monoclonal antibody 6A1. This new E. chaffeensis isolate is very similar to the Arkansas strain and provides the opportunity to substantiate the existence of diversity among ehrlichiae which infect humans. Specific factors which differ among strains may then be compared to assess their potential contributions toward cellular pathogenicity and ultimately toward the development of disease in humans.

Aged↗

Streptomycin-resistance of Euglena gracilis chloroplasts: identification of a point mutation in the 16S rRNA gene in an invariant position.

We sequenced the chloroplast 16S rRNA gene of two Euglena gracilis mutants which contain streptomycin-resistant chloroplasts (Smr 139.12/4 and Smr 139.20/2). These mutants are known to contain a single intact rrn operon per circular chloroplast genome. Nucleotide sequence comparison between a 16S rRNA gene of wild type Euglena gracilis, strain Z, with streptomycin-sensitive chloroplasts, and the 16S rRNA gene of both Smr-strains reveals a single base change (C to T) at position 876. This position is equivalent to the invariant position 912 of the E. coli 16S rRNA gene. The analogous position is also conserved in all chloroplast small subunit RNA genes from lower and higher plants sequenced so far. Light dependent protein synthesis with purified chloroplasts from streptomycin-resistant cells is not inhibited by streptomycin. Based on the results reported here we postulate linkage between the observed point mutation on the 16S rRNA gene and streptomycin-resistance of chloroplast 70S ribosomes.

Base Composition↗

Kinetic properties of rrn promoters in Escherichia coli.

How do bacteria adapt and optimize their growth in response to different environments? The answer to this question is intimately related to the control of ribosome bio-synthesis. During the last decades numerous proposals have been made to explain this control but none has been definitive. To readdress the problem, we have used measurements of rRNA synthesis rates and rrn gene dosages in E. coli to find the absolute transcription rates of the average rrn operon (transcripts per min per operon) at different growth rates. By combining these rates with lacZ expression data from rRNA promoter-lacZ fusions, the abolute activities of the isolated rrnB P1 and P2 promoters were determined as functions of the growth rate in the presence and absence of Fis and of the effector ppGpp. The promoter activity data were analyzed to obtain the relative concentrations of free RNA polymerase, [R(f)], and the ratio of the Michaelis-Menten parameters, V(max)/K(m) (promoter strength), that characterize the promoter-RNA polymerase interaction. The results indicate that changes in the basal concentration of ppGpp can account for all growth-medium dependent regulation of the rrn P1 promoter strength. The P1 promoter strength was maximal when Fis was present and the level of ppGpp was undetectable during growth in rich media or in ppGpp-deficient strains; this maximal strength was 3-fold reduced when Fis was removed and the level of ppGpp remained undetectable. At ppGpp levels above 55 pmol per cell mass unit (OD(460)) during growth in poor media, the P1 promoter strength was minimal and not affected by the presence or absence of fis. The half-maximal value occurred at 20 pmol ppGpp/OD(460) and corresponds to an intracellular concentration of about 50 microM. In connection with previously published data, the results suggest that ppGpp reduces the P1 promoter strength directly, by binding RNA polymerase, and indirectly, by inhibiting the synthesis of Fis.

DNA, Ribosomal↗

Complex promoter pattern of the single ribosomal RNA operon of an archaebacterium Halobacterium halobium.

The transcription of the single ribosomal RNA gene cluster is initiated at an assemblage of promoters five of which were previously mapped in the 910 base pair region in front of the 16S rRNA gene. The nucleotide sequence between positions -1680 and -910 was determined and S1 nuclease mapping revealed therein two sites corresponding to the 5' ends of the primary transcripts. These two presumably represent the most distal promoters, P1 and P2, of the operon, P1 being separated from the 16S rRNA gene by more than 1250 bp. An additional putative "internal" promoter located within the operon body in the 16S/23S intergenic spacer was revealed by S1 nuclease mapping. A 528 bp long open reading frame is co-transcribed with the rRNA genes from the P1 promoter. Its possible functional implication is discussed.

Base Sequence↗

Structure and regulation of the Salmonella typhimurium rnc-era-recO operon.

The Escherichia coli rnc-era-recO operon encodes ribonuclease III (RNase III; a dsRNA endonuclease involved in rRNA and mRNA processing and decay), Era (an essential G-protein of unknown functions and RecO (involved in the RecF homologous recombination pathway). Expression of the rnc and era genes is negatively autoregulated: RNase III cleaves the rncO 'operator' in the untranslated leader, destabilizing the operon mRNA. As part of a larger effort to understand RNase III and Era structure and function, we characterized rnc operon structure, function and regulation in the closely related bacterium Salmonella typhimurium. Construction of a S typhimurium strain conditionally defective for RNase III and Era expression showed that Era is essential for cell growth. This mutant strain also enabled selection of recombinant clones containing the intact S typhimurium rnc-era-recO operon, whose nucleotide sequence, predicted protein sequence, and predicted rncO RNA secondary structure were all highly conserved with those of E coli. Furthermore, genetic and biochemical analysis revealed that S typhimurium rnc gene expression is negatively autoregulated by a mechanism very similar or identical to that in E coli, and that the cleavage specificities of RNase IIIs.t. and RNase IIIE.c. are indistinguishable with regard to rncO cleavage and S typhimurium 23S rRNA fragmentation in vivo.

Bacterial Proteins↗

Isolation of kasugamycin resistant mutants in the 16 S ribosomal RNA of Escherichia coli.

Three ribosomal RNA mutations conferring resistance to the antibiotic kasugamycin were isolated using a strain of Escherichia coli in which all of the rRNA is transcribed from a plasmid-encoded rrn operon. The mutations, A794G, G926A, and A1519C, mapped to universally conserved sites in the 16 S RNA gene. Site-directed mutagenesis studies showed that virtually all mutations at these three sites conferred kasugamycin resistance and had very slight effects on cell growth. It has been known for many years that the absence of post-transcriptional modification at A1519 and the adjacent A1518 in strains lacking a functional KsgA methylase produces a kasugamycin resistance phenotype. Mutations at A1519 conferred kasugamycin resistance and had minor effects on cell growth, whereas mutations at 1518 did not confer resistance and increased the doubling time of the cells dramatically. Expression of mutations at A1518/A1519 in a methylase deficient ksgA(-)strain had divergent effects on the phenotype of the rRNA mutants, suggesting that the base identity at either position does not affect methylation at the adjacent site. Residues A794 and G926 are protected from chemical modification by kasugamycin and tRNA, and have been implicated in the initiation of protein synthesis. Despite the universal conservation and functional importance of these residues, the results presented here show that the identity of the bases is not critical for ribosomal function.

Aminoglycosides↗

The effect of trimethoprim on macromolecular synthesis in Escherichia coli. Ribosome maturation in RCstr and RCrel strains.

When Escherichia coli was inhibited with trimethoprim in media supplemented with nucleotide bases, glycine and methionine, both RC(str) and RC(rel) strains continued to accumulate RNA at rates very close to those in growing controls. The effects of trimethoprim on protein synthesis were studied by using as an experimental basis the rate of maturation of ribosomal particles from RNA-rich precursors. 1. In RC(str) cultures given nucleotide bases but no amino acids, RNA accumulation was inhibited because of amino acid lack. However, maturation of ribosomes from their precursors was more severely inhibited than was the synthesis of rRNA. The restraints on protein synthesis were more severe at the level of translation than the transcription of operons specific for the formation of ribosomal proteins. The kinetic delay time in the passage of rRNA from RNA-rich intermediates to the final ribosome products was therefore increased some three- to four-fold. 2. In RC(rel) cultures in the same conditions, trimethoprim inhibition stopped ribosomal particle synthesis, but rRNA-rich precursors accumulated. 3. If glycine+methionine were also added to inhibited RC(str) cultures, RNA accumulation resumed at a high rate. However, ribosomal maturation was still considerably disturbed because of a disproportionate response of the cells in the formation of protein and RNA. 4. With RC(rel) cultures, addition of the amino acids caused a large increase in the rate of ribosome maturation, though the degree of disproportionation between the rates of rRNA and ribosomal protein synthesis was now identical with that found in RC(str) strains. 5. When inhibited RC(rel) cultures were supplemented, there was still a severe inhibition of protein synthesis at the level of chain initiation, but inaccuracies in the process of polypeptide chain elongation were greatly decreased. This suggests that the effects of the RC(rel) mutation on the fidelity of protein synthesis in bacteria are not directed at the point of chain initiation.

Bacterial Proteins↗