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Cyclic blockade of initiation sites by streptomycin-damaged ribosomes in Escherichia coli: an explanation for dominance of sensitivity 总被引:13,自引:0,他引:13
In bacterial extracts streptomycin is known not only to inhibit ribosomal activity but also to cause gradual release of ribosomes from polysomes. Nevertheless, we now find that after streptomycin has virtually halted protein synthesis in cells of Escherichia coli K12 a substantial (though reduced) level of polysomes persists. These polysomes are evidently maintained by turnover rather than by static blockade, for in streptomycin-treated cells [3H]uracil pulses are rapidly incorporated in the polysomal messenger RNA; moreover, if the synthesis of RNA or the formylation of methionyl-transfer RNA is blocked the polysome level decreases rapidly. Streptomycin thus appears to cause a cycle of ribosomal initiation, blockage of chain extension, gradual release, and reinitiation.The resulting cyclic blockade of initiation sites can account for the dominance of streptomycin sensitivity over resistance in 2 heterozygotes. In confirmation of this model, the inactive resistant ribosomes in treated heterozygotes were found to resume activity if the cells were lysed and excess messenger was provided. These findings further suggest that in sensitive cells damage to only a fraction of the ribosomal population by streptomycin may be sufficient to block protein synthesis. 相似文献
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Summary Hybridization of cytoplasmic ribosomal RNA (rRNA) to restriction endonuclease digests of nuclear DNA of Chlamydomonas reinhardii reveals two BamHI ribosomal fragments of 2.95 and 2.35×106 d and two SalI ribosomal fragments of 3.8 and 1.5×106 d. The ribosomal DNA (rDNA) units, 5.3×106 d in size, appear to be homogeneous since no hybridization of rDNA to other nuclear DNA fragments can be detected. The two BamHI and SalI ribosomal fragments have been cloned and a restriction map of the ribosomal unit has been established. The location of the 25S, 18S and 5.8S rRNA genes has been determined by hibridizing the rRNAs to digests of the ribosomal fragments and by observing RNA/DNA duplexes in the electron microscope. The data also indicate that the rDNA units are arranged in tandem arrays. The 5S rRNA genes are not closely located to the 25S and 18S rRNA genes since they are not contained within the nuclear rDNA unit. In addition no sequence homology is detectable between the nuclear and chloroplast rDNA units of C. reinhardii.Abbreviations used rRNA
ribosomal RNA
- rDNA
ribosomal DNA d, dalton 相似文献
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BEVAN G. MILLER 《Nature: New biology》1972,237(73):109-110
OESTROGENS have been reported to stimulate preferentially the synthesis of ribosomal RNA in the castrate uterus1–4. Thus it has been suggested that 50% or more of the RNA that is synthesized in the oestrogen-stimulated uterus is ribosomal precursor RNA1,2,4. The concept is supported by the reports of enhanced ribosome formation during early oestrogen action5,6. It has also been shown, however, that during the first 6 h after oestrogen administration there is no increase in total uterine RNA in the rat uterus4,7 and also the castrate mouse uterus8. These findings seem to be incompatable with the idea that much of the RNA that is synthesized during this first 6 h is ribosomal precursor RNA, most of which accumulates as new stable rRNA. Determination of the absolute rates of total RNA synthesis in vivo should provide some insight into the amounts of various species of RNA that are synthesized after oestrogen administration. Data presented here for the rate of total RNA synthesis strongly suggest that all except a small portion of the RNA that is being synthesized at 4 h after oestrogen stimulation is unstable in vivo and hence is not ribosomal precursor RNA. 相似文献
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Frederick Chu Paul Caldwell Mark Samuels Herbert Weissbach Nathan Brot 《Biochemical and biophysical research communications》1977,76(2):593-601
The synthesis of ribosomal protein L10 has been demonstrated using λrifd18 DNA as template. The L10 synthesized forms a complex with ribosomal protein L12 and the L10 in this complex can be immunoprecipitated with L12 antiserum. 相似文献
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Ribosomal RNA Turnover in Contact Inhibited Cells 总被引:14,自引:0,他引:14
MICHAEL J. WEBER 《Nature: New biology》1972,235(54):58-61
CONTACT inhibition of animal cell growth is accompanied by a decreased rate of incorporation of nucleosides into RNA1–3. Contact inhibited cells, however, transport exogenously-supplied nucleosides more slowly than do rapidly growing cells4,5, suggesting that the rate of incorporation of isotopically labelled precursors into total cellular RNA may be a poor measure of the absolute rate of RNA synthesis by these cells. Recently, Emerson6 determined the actual rates of synthesis of ribosomal RNA (rRNA) and of the rapidly labelled heterogeneous species (HnRNA) by labelling with 3H-adenosine and measuring both the specific activity of the ATP pool and the rate of incorporation of isotope into the various RNA species. He concluded that contact inhibited cells synthesize ribosomal precursor RNA two to four times more slowly than do rapidly growing cells, but that there is little if any reduction in the instantaneous rate of synthesis of HnRNA by the non-growing cells. We have independently reached the same conclusion from simultaneous measurements on the specific radioactivity of the UTP pool and the rate of 3H-uridine incorporation into RNAs (unpublished work of Edlin and myself). However, although synthesis of the 45S precursor to ribosomal RNA is reduced two to four times in contact inhibited cells, the rate of cell multiplication and the rate of rRNA accumulation are reduced ten times. This suggests either “wastage”7 of newly synthesized 45S rRNA precursor, or turnover of ribosomes in contact inhibited cells Two lines of evidence suggest that “wastage” of 45S RNA does not play a significant role in this system. (1) The rate of synthesis of 45S RNA in both growing and contact inhibited cells agrees well with that expected from the observed rates of synthesis of 28S and 18S RNAs (unpublished work of Edlin and myself). Emerson has made similar calculations6. (2) 45S RNA labelled with a 20 min pulse of 3H-uridine is converted in the presence of actinomycin D to 28S and 18S RNAs with the same efficiency (approximately 50%) in both growing and contact inhibited cells. These results indicate that, in order to maintain a balanced complement of ribosomal RNAs, contact inhibited cells must turn over their ribosomes. We present evidence here that rRNA is stable in rapidly growing chick cells, but begins to turn over with a half-life of approximately 35–45 h as cells approach confluence and become contact inhibited. 相似文献
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Mutation in Saccharomyces cerevisiae Conferring Streptomycin and Cold Sensitivity by Affecting Ribosome Formation and Function 总被引:9,自引:3,他引:6
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A cold-sensitive, streptomycin-sensitive mutant of Saccharomyces cerevisiae accumulates a 28S ribonucleoprotein particle when grown at low temperature. This particle contains 17S ribosomal ribonculeic acid which is degraded when exposed to ribonuclease. The particle does not serve as a precursor to 60 and 40S ribosomal subunits nor is it turned over when growth is allowed to resume at the permissive temperature; rather it is only diluted by growth. That streptomycin sensitivity (allelic with cold sensitivity) is ribosomal is evidenced by the inhibition of protein synthesis in vitro by streptomycin and the binding of labeled streptomycin to the mutant but not the parental 40S ribosomal subunit. 相似文献
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Ribosomal protein S12 plays key roles in the ribosome’s response to the error-promoting antibiotic streptomycin and in modulating the accuracy of translation. The discovery that substitutions at His76 in S12, distant from the streptomycin binding site, conferred streptomycin resistance in the thermophilic bacterium Thermus thermophilus prompted us to make similar alterations in the S12 protein of Escherichia coli. While, none of the E. coli S12 mutations confers streptomycin resistance, they all have distinct effects on the accuracy of translation. In addition, a subset of the S12 alterations renders the cells hypersensitive to fusidic acid, an inhibitor of the translocation step of translation. These results indicate that the His 76 region of ribosomal protein S12 plays key roles in tRNA selection and translocation steps of protein synthesis, consistent with its interaction with elongation factors EF-Tu and EF-G, as deduced from structural studies of ribosomal complexes. 相似文献
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Ruixia Dai Jian He Xi Zha Yiting Wang Xuefei Zhang He Gao Xiaoyan Yang Juan Li Youquan Xin Yumeng Wang Sheng Li Juan Jin Qi Zhang Jixiang Bai Yao Peng Hailian Wu Qingwen Zhang Baiqing Wei Jianguo Xu Wei Li 《PLoS neglected tropical diseases》2021,15(4)
Streptomycin is considered to be one of the effective antibiotics for the treatment of plague. In order to investigate the streptomycin resistance of Y. pestis in China, we evaluated streptomycin susceptibility of 536 Y. pestis strains in China in vitro using the minimal inhibitory concentration (MIC) and screened streptomycin resistance-associated genes (strA and strB) by PCR method. A clinical Y. pestis isolate (S19960127) exhibited high-level resistance to streptomycin (the MIC was 4,096 mg/L). The strain (biovar antiqua) was isolated from a pneumonic plague outbreak in 1996 in Tibet Autonomous Region, China, belonging to the Marmota himalayana Qinghai–Tibet Plateau plague focus. In contrast to previously reported streptomycin resistance mediated by conjugative plasmids, the genome sequencing and allelic replacement experiments demonstrated that an rpsL gene (ribosomal protein S12) mutation with substitution of amino-acid 43 (K43R) was responsible for the high-level resistance to streptomycin in strain S19960127, which is consistent with the mutation reported in some streptomycin-resistant Mycobacterium tuberculosis strains. Streptomycin is used as the first-line treatment against plague in many countries. The emergence of streptomycin resistance in Y. pestis represents a critical public health problem. So streptomycin susceptibility monitoring of Y. pestis isolates should not only include plasmid-mediated resistance but also include the ribosomal protein S12 gene (rpsL) mutation, especially when treatment failure is suspected due to antibiotic resistance. 相似文献
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PROTEIN synthesis in at least some mammalian cells is probably initiated by Met-tRNAf1–3, which binds to salt-washed ribosomes at low Mg2+ concentrations in the presence of AUG and initiation factors4,5. Myosin mRNA will bind to 40S ribosomal subunits and if this represents a true initiation complex, it should bind specific initiator tRNA6,7. We report that an initiation complex specific for Met-tRNAf can be formed with the 40S ribosomal subunit isolated from mouse plasmacytoma tumours. 相似文献
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Balasubramanian Suriyanarayanan Praveena Pothuraju Lakshmi Kandasamy Dhevendaran Balakrishnan Priya Shivaani Krishna 《Journal of biomolecular structure & dynamics》2016,34(6):1190-1200
Streptomycin, an antibiotic used against microbial infections, inhibits the protein synthesis by binding to ribosomal protein S12, encoded by rpsL12 gene, and associated mutations cause streptomycin resistance. A streptomycin resistant, Lysinibacillus sphaericus DSLS5 (MIC >300 µg/mL for streptomycin), was isolated from a marine sponge (Tedania anhelans). The characterisation of rpsL12 gene showed a region having similarity to long terminal repeat sequences of murine lukemia virus which added 13 amino acids for loop formation in RpsL12; in addition, a K56R mutation which corresponds to K43R mutation present in streptomycin-resistant Escherichia coli is also present. The RpsL12 protein was modelled and compared with that of Lysinibacillus boronitolerans, Escherichia coli and Mycobacterium tuberculosis. The modelled proteins docked with streptomycin indicate compound had less affinity. The effect of loop on streptomycin resistance was analysed by constructing three different models of RpsL12 by, (i) removing both loop and mutation, (ii) removing the loop alone while retaining the mutation and (iii) without mutation having loop. The results showed that the presence of loop causes streptomycin resistance (decreases the affinity), and it further enhanced in the presence of mutation at 56th codon. Further study will help in understanding the evolution of streptomycin resistance in organisms. 相似文献
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Conformational alterations induced by streptomycin in the bacterial ribosome have been investigated using as probes, ethidium bromide, N-[14C]ethylmaleimide and a spin label nitroxide analog of N-ethylmaleimide. 1. The binding of the antibiotic to the ribosome does not affect the reactivity of sulfhydryl groups towards N-ethylmaleimide. 2. The motional freedom of spin labels bound to ribosomal proteins S1 and S18 is increased but it is hardly affected at other labeled sites. This observation suggests that the binding of streptomycin causes a local loosening of the ribosomal structure. 3. Ribosomes are found to bind less ethidium bromide in the presence of streptomycin, which suggests that the binding of streptomycin decreases the degree of organization of ribosomal RNA. 相似文献
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Tina M. Henkin Kristine M. Campbell Glenn H. Chambliss 《Molecular & general genetics : MGG》1982,186(3):347-354
Summary Revertants of a streptomycin-resistant (StrR), oligosporogenous (Spo-) mutant ofBacillus subtilis were selected for the ability to sporulate. The revertants obtained fell into two phenotypic classes: StrS Spo+ (streptomycin-sensitive, sporeforming), which arose by reversion of the streptomycin resistance mutations of the parent strain; and StrR Spo+, which arose by the acquisition of additional mutations, some of which were shown to affect ribosomal proteins. Alterations of ribosomal proteins S4 and S16 in the 30S subunit and L18 in the 50S subunit were detected in StrR Spo+ revertants by polyacrylamide gel electrophoresis. Streptomycin resistance of the parental strain and the StrR revertants was demonstrated to reside in the 30S ribosomal subunit. The second site mutations of the revertants depressed the level of streptomycin resistance in vivo and in the in vitro translation of phage SP01 messenger ribonucleic acid (mRNA) relative to the resistance exhibited by the StrR parental strain. The StrR parent grew slowly and sporulated at approximately 1% of the wild type level. The StrS revertants closely resembled the wild type strain with regard to growth and sporulation. The StrR revertants grew at rates intermediate between those of the StrR parent and wild type, and sporulated at wild type levels. 相似文献
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RNA Polymerase III of <Emphasis Type="Italic">Blastocladiella emersonii</Emphasis> is Mitochondrial 总被引:6,自引:0,他引:6
MULTIPLE RNA polymerases have been shown to exist in a wide variety of eukaryotic organisms1–5. Two nuclear polymerases have been found in all the cells studied, each with a specific location and a specific function: the DEAE fraction I enzyme is located in the nucleolus and may be involved in the synthesis of ribosomal RNA1,2,5,6; the DEAE fraction II enzyme is located in the non-nucleolar nucleoplasm and functions in the synthesis of DNA-like RNA2–5,7. The DEAE fraction III enzyme was reported to exist in sea urchin1, the aquatic fungus B. emersonii5 and to be present sometimes in rat liver preparations1,8. Although there have been some reports that polymerase III is nuclear, Horgen and Griffin5 showed that the enzyme was sensitive to the prokaryotic RNA polymerase inhibitor rifampicin. They suggested that the fraction III enzyme may be mitochondrial, formed as the result of organelle contamination in their crude nuclear preparations. The results of this study show that the DEAE fraction III enzyme in B. emersonii is a mitochondrial enzyme, most likely functioning in the synthesis of mitochondrial RNA. The rifampicin sensitivity of the enzyme is further evidence of a prokaryotic origin of mitochondria9,10. 相似文献
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《Comparative biochemistry and physiology. B, Comparative biochemistry》1978,59(2):203-206
- 1.1. The 26-S, and to a lesser extent, the 18-S ribosomal RNAs of Dendroceras reticulatum are subject to breakdown during extraction. They are stabilized by the presence of magnesium ions in the extraction medium and electrophoresis buffer.
- 2.2. The molecular weights of the undegraded ribosomal RNAs are 1.37 × 106 (26-S) and 0.67 × 106 (18-S).
- 3.3. Preliminary evidence is presented which suggests that synthesis and processing of the ribosomal RNAs occur according to the following scheme:
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We have investigated the mechanism of the expression of resistance to high levels of viomycin and coresistance to streptomycin in a mutant strain of Mycobacterium smegmatis ATCC 14468 (AC-13) which was obtained by serial transfers of parental cells to media containing increasing concentrations of viomycin. It was shown previously that resistance to viomycin by strain AC-13 was due to an alteration in the 50 S ribosomal subunit (20). However, genetic analysis has shown that mutation in 50 S subunits alone gave only low level resistance to viomycin. When a streptomycin resistant mutation (caused by an alteration in the 30 S subunit) was introduced into the low level viomycin resistant recombinant strains, most of them were highly resistant to viomycin. Some recombinants were resistant to intermediate levels of viomycin, and the remainder were not affected by the introduction of the strr allele. Studies with in vitro cell-free systems have shown that streptomycin resistant 30 S ribosomal subunits obtained from a high level viomycin resistant recombinant were able to modify the levels of resistance to viomycin expressed by the 50 S ribosomal subunit. These findings provide additional evidence concerning the functional relationship between 30 S and 50 S ribosomal components in ribosomes. 相似文献