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Initiation of Translation02:33

Initiation of Translation

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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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Aminoacyl-tRNA synthetases are present in both eukaryotes and bacteria. Though eukaryotes have 20 different aminoacyl-tRNA synthetases to couple to 20 amino acids, many bacteria do not have genes for all of these aminoacyl-tRNA synthetases. Despite this, they still use all 20 amino acids to synthesize their proteins. For instance, some bacteria do not have the gene encoding the enzyme that couples glutamine with its partner tRNA. In these organisms, one enzyme adds glutamic acid to all of the...
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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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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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Transfer RNA Synthesis02:36

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One of the unique features of tRNA is the presence of modified bases. In some tRNAs, modified bases account for nearly 20% of the total bases in the molecule. Altogether, these unusual bases protect the tRNA from enzymatic degradation by RNases.
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Transcription01:10

Transcription

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Overview
Transcription is the process of synthesizing RNA from a DNA sequence by RNA polymerase. It is the first step in producing a protein from a gene sequence. Additionally, many other proteins and regulatory sequences are involved in the proper synthesis of messenger RNA (mRNA). Regulation of transcription is responsible for the differentiation of all the different types of cells and often for the proper cellular response to environmental signals.
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Click-iT trinucleotide cap analog: Synthesis, mRNA translation, and detection.

Annamalai Senthilvelan1, Tyson Vonderfecht2, Muthian Shanmugasundaram1

  • 1Life Sciences and Laboratory Products Group, Thermo Fisher Scientific, 2130, Woodward Street, Austin, TX 78744-1832, USA.

Bioorganic & Medicinal Chemistry
|December 14, 2022
PubMed
Summary

Researchers developed a novel propargyl cap analog for mRNA. This new analog enhances translation efficiency by 1.3 times and allows for further mRNA modification via click chemistry.

Keywords:
5′-Capped RNAA549 cellCapping efficiencyClick chemistryIn vitro transcriptionTranslation efficiencyTrinucleotide cap

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Area of Science:

  • Chemical Biology
  • Molecular Biology
  • Biochemistry

Background:

  • Messenger RNA (mRNA) capping is crucial for RNA stability, export, and translation.
  • Standard cap analogs like m7G(5')ppp(5')A (m7GpppAm) are widely used in mRNA therapeutics.
  • Further modification of mRNA caps can enhance therapeutic applications.

Purpose of the Study:

  • To synthesize and characterize a novel trinucleotide cap analog containing a propargyl group: m7,3'-O-propargylG(5')PPP(5')AmG.
  • To evaluate the impact of the propargyl group on capping efficiency, in vitro transcription, and translation activity compared to a standard cap analog.
  • To explore the potential for further mRNA modification using the propargyl group via click chemistry.

Main Methods:

  • Synthesis of the novel propargyl trinucleotide cap analog.
  • In vitro transcription using T7 RNA polymerase.
  • Assessment of capping efficiency and translation activity in cultured A549 lung carcinoma epithelial cells.
  • Evaluation of mRNA modification using click chemistry.

Main Results:

  • The novel propargyl cap analog, m7,3'-O-propargylG(5')PPP(5')AmG, was successfully synthesized.
  • The propargyl cap analog functions as a substrate for T7 RNA polymerase.
  • mRNA capped with the propargyl analog demonstrated approximately 1.3-fold higher translation efficiency compared to mRNA capped with the standard GAG cap (m7G(5')ppp(5')AmG).
  • The propargyl group enables subsequent mRNA functionalization through azide-alkyne cycloaddition (click chemistry).

Conclusions:

  • The newly synthesized propargyl cap analog enhances mRNA translation efficiency.
  • The propargyl group provides a versatile handle for post-transcriptional modification of mRNA via click chemistry.
  • This propargyl cap analog holds promise for advancing mRNA-based technologies and therapeutics.