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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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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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The flow of genetic information in cells from DNA to mRNA to protein is described by the central dogma, which states that genes specify the sequence of mRNAs, which in turn specify the sequence of amino acids making up all proteins. The decoding of one molecule to another is performed by specific proteins and RNAs. Because the information stored in DNA is so central to cellular function, it makes intuitive sense that the cell would make mRNA copies of this information for protein synthesis...
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Initiation of Translation02:33

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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.
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Transcription Attenuation in Prokaryotes02:42

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Transcriptional attenuation occurs when RNA transcription is prematurely terminated due to the formation of a terminator mRNA hairpin structure.  Bacteria use these hairpins to regulate the transcription process and control the synthesis of several amino acids including histidine, lysine, threonine, and phenylalanine. Transcription attenuation takes place in the non-coding regions of mRNA.
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The Upf proteins that carry out nonsense-mediated decay (NMD) are found in all eukaryotic organisms, including humans. Each protein has an individual role, but they need to work in collaboration. Upf1 is an ATP-dependent RNA helicase that unwinds the RNA helix. Because Upf1 can unwind any RNA, Upf2 and Upf3 are required to help Upf1 discriminate between nonsense and normal mRNAs.
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Related Experiment Video

Updated: Sep 18, 2025

De novo Identification of Actively Translated Open Reading Frames with Ribosome Profiling Data
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De novo Identification of Actively Translated Open Reading Frames with Ribosome Profiling Data

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Defining the high-translational readthrough stop codon context.

Daniela Smoljanow1, Dennis Lebeda1, Julia Hofhuis1

  • 1Department for Biochemistry and Molecular Medicine, Medical School OWL, Bielefeld University, Bielefeld, Germany.

Plos Genetics
|June 25, 2025
PubMed
Summary
This summary is machine-generated.

Translational readthrough (TR) occurs when stop codons are misread. This study reveals specific DNA sequences, beyond known motifs, significantly influence TR, impacting protein production and disease therapies.

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Analysis of Termination of Transcription Using BrUTP-strand-specific Transcription Run-on TRO Approach
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Analysis of Termination of Transcription Using BrUTP-strand-specific Transcription Run-on TRO Approach

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • Translational termination is an imperfect process, often competing with elongation.
  • This inefficiency can lead to translational readthrough (TR), where near-cognate tRNAs misinterpret stop codons, causing protein C-terminal extensions.
  • The stop codon context (SCC), the surrounding nucleotide sequence, significantly influences TR rates.

Purpose of the Study:

  • To investigate the role of specific nucleotide positions within the SCC, beyond the known high-TR motif (UGA CUA G).
  • To examine how these cis-acting elements affect both basal and aminoglycoside-induced TR.
  • To understand the complex interplay of nucleotides in the expanded SCC and their impact on readthrough levels.

Main Methods:

  • Analysis of specific nucleotide positions upstream and downstream of the stop codon.
  • Examination of basal and aminoglycoside-induced translational readthrough.
  • Investigation of sequence-specific effects on TR rates.

Main Results:

  • Identified significant influence of upstream positions -9 and -8, and downstream positions +11 and +12 on readthrough levels.
  • Revealed a complex, non-linear interplay between nucleotides in the expanded SCC.
  • Demonstrated that these effects are not transferable to evolutionarily non-adapted SCCs.

Conclusions:

  • The expanded SCC plays a complex role in regulating translational readthrough.
  • Understanding these regulatory mechanisms is crucial for comprehending protein synthesis fidelity.
  • Findings may aid in developing therapies for genetic diseases caused by premature stop codon mutations.