Takayuki Konno

Publications (10)31.05 Total impact

• Article: A functional interaction of SmpB with tmRNA for determination of the resuming point of trans-translation.

  Takayuki Konno, Daisuke Kurita, Kazuma Takada, Akira Muto, Hyouta Himeno
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  ABSTRACT: 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.
  RNA 11/2007; 13(10):1723-31. · 5.09 Impact Factor
• Article: In vitro trans-translation of Thermus thermophilus: ribosomal protein S1 is not required for the early stage of trans-translation.

  Kazuma Takada, Chie Takemoto, Masahito Kawazoe, Takayuki Konno, Kyoko Hanawa-Suetsugu, Sungga Lee, Mikako Shirouzu, Shigeyuki Yokoyama, Akira Muto, Hyouta Himeno
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  ABSTRACT: 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.
  RNA 05/2007; 13(4):503-10. · 5.09 Impact Factor
• Article: [Molecular mechanism of trans-translation].

  Hyouta Himeno, Daisuke Kurita, Kazuma Takada, Takayuki Konno, Kyoko Hanawa-Suetsugu, Chie Takemoto, Masahito Kawazoe, Shigeyuki Yokoyama, Nobukazu Nameki, Gota Kawai, Akira Muto
  Seikagaku. The Journal of Japanese Biochemical Society 04/2007; 79(3):213-21. · 0.04 Impact Factor
• Article: Molecular mechanism of trans-translation.

  Daisuke Kurita, Takayuki Konno, Kazuma Takada, Akira Muto, Hyouta Himeno
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  ABSTRACT: tmRNA has a dual function as a tRNA and an mRNA to relieve the stalled ribosome. During this process, tmRNA enters the ribosomal A-site without a codonanticodon interaction, but with a protein factor SmpB. Here, we established in vitro trans-translation system, which is able to evaluate peptidyl transfer to alanyltmRNA and translation of the resume codon on tmRNA. Using this system, the effects of mutations on tmRNA or SmpB are assessed by measuring the incorporations of labeled amino acid into polypeptide. Interaction of SmpB with ribosome and tmRNA was studied by directed hydroxyl radical probing. Our results revealed that SmpB constitutes an important element during trans-translation. We propose a new model of transtranslation.
  Nucleic Acids Symposium Series 02/2007;
• Source

  Available from: PubMed Central

  Article: Competition between trans-translation and termination or elongation of translation.

  Krisana Asano, Daisuke Kurita, Kazuma Takada, Takayuki Konno, Akira Muto, Hyouta Himeno
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  ABSTRACT: The effects of tRNA, RF1 and RRF on trans-translation by tmRNA were examined using a stalled complex of ribosome prepared using a synthetic mRNA and pure Escherichia coli translation factors. No endoribonucleolytic cleavage of mRNA around the A site was found in the stalled ribosome and was required for the tmRNA action. When the A site was occupied by a stop codon, alanyl-tmRNA competed with RF1 with the efficiency of peptidyl-transfer to alanyl-tmRNA for trans-translation inversely correlated to the efficiency of translation termination. The competition was not affected by RF3. A sense codon also serves as a target for alanyl-tmRNA with competition of aminoacyl-tRNA. The extent of inhibition was decreased with the length of the 3'-extension of mRNA. RRF, only at a high concentration, slightly affected peptidyl-transfer for trans-translation, although it did not affect the canonical elongation. These results indicate that alanyl-tmRNA does not absolutely require the truncation of mRNA around the A site but prefers an mRNA of a short 3'-extension from the A site and that it can operate on either a sense or termination codon at the A site, at which alanyl-tmRNA competes with aminoacyl-tRNA, RF and RRF.
  Nucleic Acids Research 02/2005; 33(17):5544-52. · 8.03 Impact Factor
• Article: Functional analysis of factors involved in trans-translation.

  Kazuma Takada, Chie Takemoto, Masahito Kawazoe, Takayuki Konno, Noriko Matsuta, Daisuke Kurita, Krisana Asano, Mikako Shirouzu, Shigeyuki Yokoyama, Akira Muto, Hyouta Himeno
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  ABSTRACT: trans-translation is a rescue system for stalled ribosomes to terminate the translation for recycling of ribosome and lead the incomplete protein on the stalled ribosome to degradation. In trans-translation, several proteins and RNAs are involved. tmRNA and SmpB are only factors known to be essential for trans-translation. EF-Tu and EF-G functions in translation and also are seemed to function in trans-translation. Ribosomal protein S1 is known to bind tmRNA. In this study, we cloned the gene of tmRNA from Thermus thermophilus and analyzed the functions of factors involved in trans-translation in vitro.
  Nucleic Acids Symposium Series 02/2005;
• Article: Initiation-shift of trans-translation by aminoglycosides.

  Takayuki Konno, Daisuke Kurita, Toshiharu Takahashi, Akira Muto, Hyouta Himeno
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  ABSTRACT: Trans-translation is an unusual translation in which tmRNA plays a dual function as a tRNA and an mRNA to relieve the stalled translation on the ribosome. In this study, we examined the effects of several kinds of aminoglycoside antibiotics, on trans-translation in vitro. A chemical footprinting study revealed that paromomycins bind tmRNA in the tRNA domain and in the middle of the long helix between tRNA and mRNA domains. Paromomycin bound in the tRNA domain inhibited aminoacylation, and the inhibition was suppressed by the addition of SmpB, a tmRNA binding protein. It was also found that paromomycin causes a shift of the translation-resuming point on tmRNA by -1. The effect on initiation-shift was suppressed by a mutation at the paromomycin binding site in 16S rRNA, but not by mutations in tmRNA. The effect of paromomycin on trans-translation differs substantially from that on canonical translation, in which it induces miscoding by modulating the A site of the decoding helix of the small subunit RNA of the ribosome. A minimal structure that causes initiation-shift was identified.
  Nucleic Acids Symposium Series 02/2004;
• Source

  Available from: ncbi.nlm.nih.gov

  Article: A minimum structure of aminoglycosides that causes an initiation shift of trans-translation.

  Takayuki Konno, Toshiharu Takahashi, Daisuke Kurita, Akira Muto, Hyouta Himeno
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  ABSTRACT: Trans-translation is an unusual translation in which transfer-messenger RNA plays a dual function--as a tRNA and an mRNA--to relieve the stalled translation on the ribosome. It has been shown that paromomycin, a typical member of a 4,5-disubstituted class of aminoglycosides, causes a shift of the translation-resuming point on the tmRNA by -1 during trans-translation. To address the molecular basis of this novel effect, we examined the effects of various aminoglycosides that can bind around the A site of the small subunit of the ribosome on trans-translation in vitro. Tobramycin and gentamicin, belonging to the 4,6-disubstituted class of aminoglycosides having rings I and II similar to those in the 4,5-disubstituted class, possess similar effects. Neamine, which has only rings I and II, a common structure shared by 4,5- and 4,6-disubstituted classes of aminoglycosides, was sufficient to cause an initiation shift of trans-translation. In contrast, streptomycin or hygromycin B, lacking ring I, did not cause an initiation shift. The effect of each aminoglycoside on trans-translation coincides with that on conformational change in the A site of the small subunit of the ribosome revealed by recent structural studies: paromomycin, tobramycin and geneticin which is categorized into the gentamicin subclass, but not streptomycin and hygromycin B, flip out two conserved adenine bases at 1492 and 1493 from the A site helix. The pattern of initiation shifts by paromomycin fluctuates with variation of mutations introduced into a region upstream of the initiation point.
  Nucleic Acids Research 02/2004; 32(14):4119-26. · 8.03 Impact Factor
• Article: Various effects of paromomycin on tmRNA-directed trans-translation.

  Toshiharu Takahashi, Takayuki Konno, Akira Muto, Hyouta Himeno
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  ABSTRACT: trans-Translation is an unusual translation in which tmRNA plays a dual function as a tRNA and an mRNA to relieve the stalled translation on the ribosome. In this study, we examined the effects of an aminoglycoside antibiotic, paromomycin, on several tmRNA-related events in vitro. The results of a chemical footprinting study indicated that paromomycin molecules bind tmRNA at G332/G333 in the tRNA domain and A316 in the middle of the long helix between tRNA and mRNA domains. Paromomycin bound at G332/G333 inhibited aminoacylation, and the inhibition was suppressed by the addition of SmpB, a tmRNA-binding protein. It was also found that paromomycin causes a shift of the translation resuming point on tmRNA by -1. The effect on initiation shift was canceled by a mutation at the paromomycin-binding site in 16 S rRNA but not by mutations in tmRNA. A high concentration of paromomycin inhibited trans-translation, whereas it enhanced the initiation-shifted trans-translation when SmpB was exogenously added or a mutation was introduced at 333. The effect of paromomycin on trans-translation differs substantially from that on canonical translation, in which it induces miscoding by modulating the A site of the decoding helix of the small subunit RNA of the ribosome.
  Journal of Biological Chemistry 08/2003; 278(30):27672-80. · 4.77 Impact Factor
• Article: Various effects of paromomycin on tmRNA-mediated trans-translation.

  Takayuki Konno, Toshiharu Takahashi, Akira Muto, Hyouta Himeno
  [show abstract] [hide abstract]
  ABSTRACT: Trans-translation is an unusual translation in which tmRNA plays a dual function as a tRNA and an mRNA to relieve the stalled translation on the ribosome. In this study, we examined the effects of an aminoglycoside antibiotic, paromomycin, on several tmRNA-related events in vitro. A chemical footprinting study revealed that paromomycins bind tmRNA in the tRNA domain and in the middle of the long helix between tRNA and mRNA domains. Paromomycin bound in the tRNA domain inhibited aminoacylation, and the inhibition was suppressed by the addition of SmpB, a tmRNA binding protein. It was also found that paromomycin causes a shift of the translation-resuming point on tmRNA by -1. The effect on initiation-shift was canceled by a mutation at the paromomycin binding site in 16S rRNA, but not by mutations in tmRNA. The effect of paromomycin on trans-translation differs substantially from that on canonical translation, in which it induces miscoding by modulating the A site of the decoding helix of the small subunit RNA of the ribosome.
  Nucleic acids research. Supplement (2001). 02/2003;