RNA folding kinetics regulates translation of phage MS2 maturation gene

^*Leiden Institute of Chemistry, Department of Biochemistry, Gorlaeus Laboratories, University of Leiden, P.O. Box 9502, 2300 RA Leiden, The Netherlands; and ^‡Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Ulicȧ Miklucho–Maklaya 16/10, 117871 Moscow, Russia

Edited by Peter H. von Hippel, University of Oregon, Eugene, OR, and approved July 8, 1997 (received for review January 27, 1997)

Abstract

The gene for the maturation protein of the single-stranded RNA coliphage MS2 is preceded by an untranslated leader of 130 nt, which folds into a cloverleaf, i.e., three stem–loop structures enclosed by a long distance interaction (LDI). This LDI prevents translation because its 3′ moiety contains the Shine–Dalgarno sequence of the maturation gene. Previously, several observations suggested that folding of the cloverleaf is kinetically delayed, providing a time window for ribosomes to access the RNA. Here we present direct evidence for this model. In vitro experiments show that ribosome binding to the maturation gene is faster than refolding of the denatured cloverleaf. This folding delay appears related to special properties of the leader sequence. We have replaced the three stem–loop structures by a single five nt loop. This change does not affect the equilibrium structure of the LDI. Nevertheless, in this construct, the folding delay has virtually disappeared, suggesting that now the RNA folds faster than ribosomes can bind. Perturbation of the cloverleaf by an insertion makes the maturation start permanently accessible. A pseudorevertant that evolved from an infectious clone carrying the insertion had overcome this defect. It showed a wild-type folding delay before closing down the maturation gene. This experiment reveals the biological significance of retarded cloverleaf formation.

Footnotes

↵ † Present address: European Molecular Biology Laboratory, D6g Heidelberg, Germany.
↵ § To whom reprint requests should be addressed at: Mathematics and Natural Sciences, Leiden Institute of Chemistry, Department of Biochemistry, Einsteinweg 55, 2300 RA Leiden, The Netherlands. e-mail: duin_j{at}rulgca.leidenuniv.nl.
This paper was submitted directly (Track II) to the Proceedings office. Abbreviations: LDI, long distance interaction; RBS, ribosome binding site.