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Protein S1 counteracts the inhibitory effect of the extended Shine-Dalgarno sequence on translation
Anastassia V Komarova1, Ludmila S Tchufistova, Elena V Supina
1Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow.
Abstract:
There are two major components of Escherichia coli ribosomes directly involved in selection and binding of mRNA during initiation of protein synthesis-the highly conserved 3' end of 16S rRNA (aSD) complementary to the Shine-Dalgarno (SD) domain of mRNA, and the ribosomal protein S1. A contribution of the SD-aSD and S1-mRNA interactions to translation yield in vivo has been evaluated in a genetic system developed to compare efficiencies of various ribosome-binding sites (RBS) in driving beta-galactosidase synthesis from the single-copy (chromosomal) lacZ gene. The in vivo experiments have been supplemented by in vitro toeprinting and gel-mobility shift assays. A shortening of a potential SD-aSD duplex from 10 to 8 and to 6 bp increased the beta-galactosidase yield (four- and sixfold, respectively) suggesting that an extended SD-aSD duplex adversely affects translation, most likely due to its redundant stability causing ribosome stalling at the initiation step. Translation yields were significantly increased upon insertion of the A/U-rich S1 binding targets upstream of the SD region, but the longest SD remained relatively less efficient. In contrast to complete 30S ribosomes, the S1-depleted 30S particles have been able to form an extended SD-aSD duplex, but not the true ternary initiation complex. Taken together, the in vivo and in vitro data allow us to conclude that S1 plays two roles in translation initiation: It forms an essential part of the mRNA-binding track even when mRNA bears a long SD sequence, and through the binding to the 5' untranslated region, it can ensure a substantial enhancing effect on translation.
Insights
The Shine-Dalgarno (SD) sequence and 16S rRNA (aSD) interaction in Escherichia coli translation initiation is optimized by shorter duplexes. Ribosomal protein S1 enhances translation yield by binding mRNA upstream of the SD region.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Protein synthesis initiation in Escherichia coli involves interactions between the 16S rRNA anti-Shine-Dalgarno (aSD) sequence and the mRNA Shine-Dalgarno (SD) domain.
- Ribosomal protein S1 also plays a role in mRNA binding and translation initiation.
Purpose of the Study:
- To evaluate the contribution of SD-aSD and S1-mRNA interactions to translation yield in vivo.
- To investigate the impact of Shine-Dalgarno duplex length and S1 binding on translation efficiency.
Main Methods:
- Development of a genetic system to compare ribosome-binding site efficiencies for beta-galactosidase synthesis.
- In vivo experiments complemented by in vitro toeprinting and gel-mobility shift assays.
Main Results:
- Shortening the SD-aSD duplex from 10 to 6 base pairs increased beta-galactosidase yield four- to sixfold, indicating that extended duplexes hinder translation.
- Insertion of A/U-rich S1 binding targets upstream of the SD region significantly increased translation yields.
- S1-depleted 30S ribosomal particles could form extended SD-aSD duplexes but not true ternary initiation complexes.
Conclusions:
- An extended Shine-Dalgarno duplex adversely affects translation initiation, likely due to excessive stability causing ribosome stalling.
- Ribosomal protein S1 has two key roles: it facilitates mRNA binding, even with long SD sequences, and enhances translation by binding the 5' untranslated region.