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Nucleotide Context Can Modulate Promoter Strength in Genes Transcribed by RNA Polymerase III.

Danil V Stasenko1, Karina A Tatosyan1, Olga R Borodulina1

  • 1Laboratory of Eukaryotic Genome Evolution, Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 119991 Moscow, Russia.

Genes
|April 28, 2023
PubMed
Summary

Small nuclear RNAs 4.5SH and 4.5SI genes have distinct promoter elements. Surprisingly, swapping these elements between genes unexpectedly reduced promoter activity, highlighting the importance of the nucleotide environment for RNA polymerase III transcription strength.

Keywords:
Mus musculusRNA polymerase IIISINEnon-coding RNApromotortranscriptiontransfection

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • Small nuclear RNAs (snRNAs) are crucial for various cellular processes.
  • The genes encoding 4.5SH and 4.5SI RNAs, originating from 7SL RNA and tRNA respectively, are found in rodents.
  • These genes utilize RNA polymerase III (pol III) for transcription and possess characteristic promoter elements (Boxes A, B, and TATA-like).

Purpose of the Study:

  • To investigate the functional roles of specific promoter elements (Boxes A, B, TATA-like) in the transcription of 4.5SH and 4.5SI RNA genes.
  • To compare the promoter strengths of 4.5SH and 4.5SI RNA genes.
  • To determine the impact of the nucleotide environment on pol III-directed promoter activity.

Main Methods:

  • Site-directed mutagenesis was used to swap promoter elements between 4.5SH and 4.5SI RNA genes.
  • Transfection of modified gene constructs into HeLa cells to assess transcription levels.
  • Development of a novel competitive co-transfection assay to quantitatively compare promoter strengths.

Main Results:

  • Simultaneous replacement of all three promoter boxes in the 4.5SH gene with those from the 4.5SI gene resulted in a 40% decrease in transcription.
  • The developed competitive assay revealed that the 4.5SI promoter is 12 times stronger than the 4.5SH promoter.
  • Unexpectedly, the transfer of strong promoter elements from 4.5SI to the 4.5SH gene significantly diminished promoter activity.

Conclusions:

  • The nucleotide environment surrounding promoter elements plays a critical role in determining the strength of RNA polymerase III-directed promoters.
  • Promoter element function is context-dependent, and simple element swapping may not yield predictable outcomes.
  • Further research is needed to elucidate the mechanisms by which the nucleotide environment influences pol III transcription initiation and efficiency.