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In trans-translation, transfer-messenger RNA (tmRNA), possessing a dual function as a tRNA and an mRNA, relieves a stalled translation on the ribosome with the help of SmpB. Here, we established an in vitro system using Escherichia coli translation and trans-translation factors to evaluate two steps of trans-translation, peptidyl transfer from peptidyl-tRNA to alanyl-tmRNA and translation of the resume codon on tmRNA. Using this system, the effects of several mutations upstream of the tag-encoding region on tmRNA were examined. These mutations affected translation of the resume codon rather than peptidyl transfer, and one of them, A84U/U85G, caused a shift of the resume codon by -1. We also found that U(85) is protected from chemical modification by SmpB. In the A84U/U85G mutant, the base of protection was shifted from 85 to 84. Another mutation, A86U, which caused a shift of the resume codon by +1, shifted the base of protection from 85 to 86. The protection at 85 was suppressed by a mutation in the tRNA-like domain critical to SmpB binding. These results suggest that SmpB serves to bridge two separate domains of tmRNA to determine the initial codon for tag-translation. A mutant SmpB with a truncation of the unstructured C-terminal tail failed to promote peptidyl transfer, although it still protected U(85) from chemical modification. 相似文献
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In vitro trans-translation of Thermus thermophilus: ribosomal protein S1 is not required for the early stage of trans-translation 下载免费PDF全文
Takada K Takemoto C Kawazoe M Konno T Hanawa-Suetsugu K Lee S Shirouzu M Yokoyama S Muto A Himeno H 《RNA (New York, N.Y.)》2007,13(4):503-510
Transfer-messenger RNA (tmRNA) plays a dual role as a tRNA and an mRNA in trans-translation, during which the ribosome replaces mRNA with tmRNA encoding the tag-peptide. These processes have been suggested to involve several tmRNA-binding proteins, including SmpB and ribosomal protein S1. To investigate the molecular mechanism of trans-translation, we developed in vitro systems using purified ribosome, elongation factors, tmRNA and SmpB from Thermus thermophilus. A stalled ribosome in complex with polyphenylalanyl-tRNA(Phe) was prepared as a target of tmRNA. A peptidyl transfer reaction from polyphenylalanyl-tRNA(Phe) to alanyl-tmRNA was observed in an SmpB-dependent manner. The next peptidyl transfer to aminoacyl-tRNA occurred specifically to the putative resume codon for the tag-peptide, which was confirmed by introducing a mutation in the codon. Thus, the in vitro systems developed in this study are useful to investigate the early steps of trans-translation. Using these in vitro systems, we investigated the function of ribosomal protein S1, which has been believed to play a role in trans-translation. Although T. thermophilus S1 tightly bound to tmRNA, as in the case of Escherichia coli S1, it had little or no effect on the early steps of trans-translation. 相似文献
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Ko-ichiro Miyamoto Parida Yamada Ryo-taro Yamaguchi Takami Muto Ayumi Hirano Yasuo Kimura Michio Niwano Hiroko Isoda 《Cytotechnology》2007,55(2-3):143-149
In this study, we report on an in situ monitoring system of living cultured cells using infrared absorption spectroscopy in
the geometry of multiple internal reflections (MIR-IRAS). In order to observe living cultured cells, the temperature in the
sample chamber of a FT-IR spectrometer was maintained at 37 °C and a humidified gas mixture containing 5% CO2 was introduced into the sample chamber. Human breast cell line MCF-7 cultured on Si MIR prisms were placed in the sample
chamber and infrared spectra of MCF-7 cells were collected for 5 h. It was found that the adhesion and metabolism of MCF-7
cells could be monitored by the absorption intensity of amide-II protein band (1,545 cm−1) and also by the absorption intensities of CH
x
bands (2,700–3,100 cm−1). These results suggest that our system is useful for a nondestructive and non-label monitoring of cell viability. Our method
based on infrared absorption spectroscopy has a potential for bioscreening application. 相似文献
65.
Ujifuku Kenta Fujimoto Takashi Sato Kei Morofuji Yoichi Muto Hideki Masumoto Hiroshi Nakagawa Shinsuke Niwa Masami Matsuo Takayuki 《Cellular and molecular neurobiology》2022,42(4):997-1004
Cellular and Molecular Neurobiology - Metastatic brain tumors have poor prognoses and pose unmet clinical problems for the patients. The blood–brain barrier (BBB) implication is supposed to... 相似文献
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Wakui S Motohashi M Muto T Takahashi H Hano H Jutabha P Anzai N Wempe MF Endou H 《Comparative medicine》2011,61(5):412-418
Epidemiologic studies indicate that the incidence of gastric cancer is higher in males than in females. Although the mechanisms mediating this difference are unclear, a role for estrogens has been proposed. We used Western blotting to evaluate the role of estrogen receptor (ER) subtypes ERα and ERβ and proliferating cell nuclear antigen (PCNA) in N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced gastric carcinogenesis in Wistar rats; ERα and ERβ mRNA levels also were analyzed by quantitative real-time RT-PCR analysis. The incidence of gastric cancer was significantly higher in male than female rats. In both sexes, ERα expression was similar in MNNG-treated cancerous and noncancerous tissues and normal gastric tissue. However, ERβ expression in MNNG-treated cancerous and noncancerous tissues was significantly lower in male rats and higher in female rats than that in normal gastric tissue; MNNG-induced cancerous tissue showed the highest ERβ expression. PCNA expression in MNNG-treated cancerous tissues was higher than that in noncancerous tissues, and was higher in male rats than female rats. Western blotting results were consistent with the mRNA changes determined by quantitative real-time RT-PCR. The present study provides evidence of a sex-associated difference in ERβ and PCNA expression in MNNG-induced gastric cancers in Wistar rats. 相似文献
69.
Tetsuji Nakabo Kouji Nakayama Nozomu Muto Masayuki Miyazawa 《Ichthyological Research》2011,58(2):180-183
Oncorhynchus kawamurae (Osteichthyes: Salmonidae) (common name “Kunimasu”), a species endemic to Lake Tazawa, Akita Prefecture, Japan, was believed
to have been extinct since 1940. However, nine specimens were discovered in March and April 2010 in Lake Saiko, Yamanashi
Prefecture, one of the lakes to which eyed eggs of the species were introduced in 1935. These were identified as O. kawamurae because of having 47–62 pyloric caeca, 37–43 gill-rakers, a black-colored body, and spawning at 30–40 m depth in early spring,
which are unique characteristics within Oncorhynchus. Furthermore, the distinctiveness of Kunimasu from sympatric kokanee (O. nerka) was supported by microsatellite DNA data. 相似文献
70.