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Dual DNA Rulers to Study the Mechanism of Ribosome Translocation with Single-Nucleotide Resolution
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Extending the Spacing between the Shine-Dalgarno Sequence and P-Site Codon Reduces the Rate of mRNA Translocation

Hironao Wakabayashi1, Chandani Warnasooriya1, Dmitri N Ermolenko1

  • 1Department of Biochemistry & Biophysics at School of Medicine and Dentistry and Center for RNA Biology, University of Rochester, Rochester, NY 14642, USA.

Journal of Molecular Biology
|June 17, 2020
PubMed

Insights

The spacer length between the Shine-Dalgarno sequence and the P-site codon impacts translation. Longer spacers significantly slow ribosome translocation and promote mRNA frameshifting during protein synthesis.

Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • Shine-Dalgarno (SD) sequences at the 5' end of mRNA are crucial for translation initiation by binding to 16S rRNA.
  • Intragenic SD-like sequences can influence ribosome behavior during protein synthesis, including frameshifting and translocation speed.

Purpose of the Study:

  • To investigate the effect of the spacer length between the Shine-Dalgarno (SD) sequence and the P-site codon on ribosome translocation rates.
  • To determine how spacer length influences mRNA-ribosome interactions and frameshifting during translation elongation.

Main Methods:

  • Utilized in vitro translation systems to measure ribosome translocation rates.
  • Manipulated the length of the spacer sequence between the SD element and the start codon in mRNA constructs.
  • Analyzed mRNA-tRNA-ribosome complex stability and frameshifting efficiency.

Main Results:

  • The length of the spacer between the SD sequence and the P-site codon significantly affects ribosome translocation.
  • Increasing the spacer length beyond 6 nucleotides destabilizes mRNA-tRNA-ribosome interactions.
  • A spacer length greater than 6 nt leads to a 5- to 10-fold reduction in the rate of ribosome translocation.

Conclusions:

  • Spacer length is a critical determinant of translation elongation speed and fidelity.
  • Structural rearrangements within the spacer region likely impede ribosome movement and promote frameshifting.
  • Findings provide insights into the regulation of translation and frameshifting mechanisms.
Keywords:
Förster resonance energy transferframeshiftingtoeprinting assaytranslation pausingtranslocation kinetics

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