SUMMARY
Evolution and essential features of mRNA translation initiation regions in
prokaryotes. An analysis of phage MS2 RNA.
Normunds Līcis
The form of the thesis is the summary of published or prepared for publication scientific articles. The work
was carried out in Biomedical Research and Study Centre of Latvian University in the period between 1991
and 1997 years and some of the experiments were done in Gorlaeus Laboratories of Leiden University, The
Netherlands.
Gene expression in prokaryotes ultimately depends on the efficiency and the accuracy of
translation that in turn is determined at the stage when ribosomes recognize and bind to
mRNA translation initiation region (TIR). The properties of TIR are the major determinant
of the efficiency of translation initiation and their revealing is of importance both from
theoretical and practical viewpoint, for example application in biotechnology. The present
work is devoted to both aspects of general relevance for control of prokaryotic translation by
features of mRNA TIR and particular molecular basis of translational control in the subjects
of this study - single stranded RNA bacteriophages MS2 and fr. Phage MS2 is a member of
a group of bacterial viruses in which the genetic information is stored in and expressed
directly from RNA genome and thus the gene expression in these organisms is controlled
mainly at the translational level. Two general approaches have been used in the present
work. The first part of the thesis concerns with in vitro experiments whereas in the second
part we have exploited a characteristic feature of most RNA viruses - a high adaptability - to
test the phage RNA properties essential for translational control by natural evolution. In
vitro data allowed us to conclude that all the important information for an efficient initiation
of the replicase gene translation in phage MS2 and its close relative phage fr TIR does
not expand outside a short RNA fragment in length of 16 nucleotides. The results also
imply that within this minimal active initiation region only the translation initiation codon
AUG and a 5'-region containing the so-called SD-sequence are important for the efficiency
of the initiation process. The results of evolutionary studies on phage MS2 coat protein gene
and replicase protein gene TIRs show that apart SD-sequence and initiation codon there are
no other sequence-specific features of a general relevance for the efficiency of translation
initiation. At the same time an essential property of an initiation site was found to be the
strength of secondary structure that occludes TIR and may exert strong negative effect on the
initiation rate. It was also shown, in agreement with earlier suggestions, that the global
phage RNA folding is important for the control of the replicase gene translation. We
observed as well that the extent of suppression is strongly dependent on sequence/structure
of the region involving the replicase gene start or its surroundings. Our data also show that
although SD-sequence is essential for the translation initiation it is not an absolute
requirement for ribosome binding to take place
The results of the this work are compiled in 5 articles, 3 of which have been published
but 2 are prepared for publication in ISI journals, and also presented in 6 international
congresses and conferences.