Four codons in the cat-86 leader define a chloramphenicol-sensitive ribosome stall sequence

Area of Science:

• Bacterial genetics
• Molecular biology
• Gene regulation

Background:

• Chloramphenicol acetyltransferase (cat) genes in gram-positive bacteria are induced by chloramphenicol.
• This induction is mediated by ribosome stalling at a specific site in the cat leader mRNA.
• Ribosome stalling alters downstream RNA secondary structure, revealing the ribosome-binding site for the cat coding sequence.

Purpose of the Study:

• To investigate the role of specific leader codons in ribosome stalling within the cat-86 leader mRNA.
• To determine the contribution of the stall sequence's position and its interaction with ribosomal RNA to cat gene induction.

Main Methods:

• Analysis of ribosome stalling in cat-86 leader mRNA based on leader codons 2-5.
• Experimental manipulation of the stall sequence's position relative to the authentic stall sequence.
• Investigation of complementarity between the cat leader sequence and Bacillus subtilis 16S ribosomal RNA.

Main Results:

• Ribosome stalling in the cat-86 leader is dependent on leader codons 2-5 (Val-Lys-Thr-Asp) and requires the correct reading frame.
• Displacing the stall sequence reduced cat-86 induction, indicating its function is not solely dependent on proximity to downstream structures.
• A region within the stall sequence exhibits complementarity to Bacillus subtilis 16S rRNA, which is absent in Escherichia coli.

Conclusions:

• The tetrapeptide specified by codons 2-5 plays an active role in chloramphenicol induction.
• Complementarity between the leader mRNA and 16S rRNA may facilitate ribosome positioning or potentiate stalling, contributing to differential induction in various bacterial species.
• The findings elucidate a novel mechanism of translational control in bacterial gene expression.