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Related Concept Videos

Leaky Scanning02:28

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During most eukaryotic translation processes, the small 40S ribosome subunit scans an mRNA from its 5' end until it encounters the first start AUG codon. The large 60S ribosomal subunit then joins the smaller one to initiate protein synthesis. The location of the translation initiation is largely determined by the nucleotides near the start codon as there may be multiple translation initiation sites present on the mRNA.  Marilyn Kozak discovered that the sequence RCCAUGG (where R...
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Initiating translation is complex because it involves multiple molecules. Initiator tRNA, ribosomal subunits, and eukaryotic initiation factors (eIFs) are all required to assemble on the initiation codon of mRNA. This process consists of several steps that are mediated by different eIFs.
First, the initiator tRNA must be selected from the pool of elongator tRNAs by eukaryotic initiation factor 2 (eIF2). The initiator tRNA (Met-tRNAi) has conserved sequence elements including modified bases at...
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Base complementarity between the three base pairs of mRNA codon and the tRNA anticodon is not a failsafe mechanism. Inaccuracies can range from a single mismatch to no correct base pairing at all. The free energy difference between the correct and nearly correct base pairs can be as small as 3 kcal/ mol. With complementarity being the only proofreading step, the estimated error frequency would be one wrong amino acid in every 100 amino acids incorporated. However, error frequencies observed in...
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Ribosomes01:27

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Ribosomes translate genetic information encoded by messenger RNA (mRNA) into proteins. Both prokaryotic and eukaryotic cells have ribosomes. Cells that synthesize large quantities of protein—such as secretory cells in the human pancreas—can contain millions of ribosomes.
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In eukaryotes, transcription and translation are compartmentalized; an mRNA is first synthesized in the nucleus and then selectively transported to the cytoplasm for protein synthesis. Before transport, a pre-mRNA undergoes several steps of post-transcriptional modifications including splicing, 5' capping, and the addition of a poly-adenine tail. Various proteins bind to the pre-mRNA during these modifications. The mRNA transport takes place with the help of multiple proteins playing...
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Termination of Translation01:44

Termination of Translation

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The large ribosomal subunit has several important structures essential to translation. These include the peptidyl transferase center (PTC) - which is the site where the peptide bond is formed - and a large, internal, water-filled tube through which the nascent polypeptide moves. This latter structure is called the Peptide Exit Tunnel, and it begins at the PTC and spans the body of the large ribosomal subunit. During translation, as the nascent polypeptide chain is synthesized, it passes through...
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40S ribosomal subunits scan mRNA for the start codon by one-dimensional diffusion.

Hironao Wakabayashi1, Mingyi Zhu1, Elizabeth J Grayhack1

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

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mRNA scanning by the 40S ribosomal subunit in yeast is primarily driven by one-dimensional diffusion, not helicase activity. Unstructured 5' untranslated regions (UTRs) have minimal impact, while secondary structures significantly inhibit translation.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • Eukaryotic translation initiation involves the 40S ribosomal subunit scanning the 5' untranslated region (UTR) of mRNA to find the start codon.
  • The precise molecular mechanism governing mRNA scanning remains incompletely understood.

Purpose of the Study:

  • To investigate the molecular mechanism of mRNA scanning by the 40S ribosomal subunit during eukaryotic translation initiation.
  • To determine the roles of RNA helicases and other initiation factors in mRNA scanning and their impact on protein synthesis.

Main Methods:

  • Utilized Green Fluorescent Protein (GFP) reporters in Saccharomyces cerevisiae (yeast) cells.
  • Assessed the effect of varying unstructured and structured 5' UTR lengths on protein synthesis.
  • Introduced loss-of-function mutations in key translational RNA helicases (eIF4A, Ded1) and initiation factors (eIF4G, eIF4B, eIF3g, eIF3i).

Main Results:

  • Order-of-magnitude changes in unstructured 5' UTR lengths had a modest effect on protein synthesis, suggesting scanning is not rate-limiting.
  • Secondary structures within the 5' UTR significantly inhibited translation.
  • Mutations in RNA helicases and initiation factors reduced translation, particularly for structured 5' UTRs, but helicases were not rate-limiting for 40S movement.

Conclusions:

  • One-dimensional diffusion, rather than helicase-driven translocation, predominantly drives 40S subunit scanning along the mRNA 5' UTR in yeast.
  • RNA helicases eIF4A and Ded1 are crucial for 40S recruitment and unwinding secondary structures but do not limit the scanning rate.