# Role of Ribosomal Protein bS1 in Orthogonal mRNA Start Codon Selection

## TL;DR

This study investigates how the ribosomal protein bS1 and the Shine-Dalgarno sequence influence the selection of mRNA start codons in bacteria.

## Contribution

The study shows that removing bS1 from ribosomes does not improve their orthogonality, suggesting other mRNA features are involved.

## Key findings

- Ribosomes lacking bS1 are slightly less active than wild-type ribosomes in vitro.
- Orthogonal ribosomes without bS1 do not show improved orthogonality.
- mRNA features beyond the Shine-Dalgarno sequence and bS1 binding likely affect start codon selection.

## Abstract

In many bacteria, the location of the mRNA start codon
is determined
by a short ribosome binding site sequence that base pairs with the
3′-end of 16S rRNA (rRNA) in the 30S subunit. Many groups have
changed these short sequences, termed the Shine–Dalgarno (SD)
sequence in the mRNA and the anti-Shine–Dalgarno (ASD) sequence
in 16S rRNA, to create “orthogonal” ribosomes to enable
the synthesis of orthogonal polymers in the presence of the endogenous
translation machinery. However, orthogonal ribosomes are prone to
SD-independent translation. Ribosomal protein bS1, which binds to
the 30S ribosomal subunit, is thought to promote translation initiation
by shuttling the mRNA to the ribosome. Thus, a better understanding
of how the SD and bS1 contribute to start codon selection could help
efforts to improve the orthogonality of ribosomes. Here, we engineered
the Escherichia coli ribosome to prevent binding of bS1 to the 30S subunit and separate
the activity of bS1 binding to the ribosome from the role of the mRNA
SD sequence in start codon selection. We find that ribosomes lacking
bS1 are slightly less active than wild-type ribosomes in vitro. Furthermore,
orthogonal 30S subunits lacking bS1 do not have an improved orthogonality.
Our findings suggest that mRNA features outside the SD sequence and
independent of binding of bS1 to the ribosome likely contribute to
start codon selection and the lack of orthogonality of present orthogonal
ribosomes.