Influences on translation initiation and early elongation by the messenger RNA region flanking the initiation codon at the 3′ side
Introduction
The initiation process and the formation of the initiation complex determine the efficiency of gene expression at the translational level. The sequences surrounding the start triplet act as determinants, influencing this efficiency (Dreyfus, 1988, Stenström et al., 2001b and references therein). The Shine-Dalgarno sequence (SD), a few bases upstream of the initiation codon is complementary to the anti-Shine-Dalgarno sequence near the 3′ end of the 16S rRNA (Shine and Dalgarno, 1974, O'Connor et al., 1999 and references therein; Stenström et al., 2001a and references therein). The purine-rich SD region is of prime importance for the association of messenger RNA (mRNA) to the 30S ribosomal subunit, thereby increasing the probability for decoding of the initiation codon (AUG, GUG, UUG and in one case AUU). The complementary base pairing between the SD and the anti-SD in the 16S rRNA during an early initiation step has been well established by site directed mutagenesis (O'Connor et al., 1999 and references therein).
Even though the initiation codon and the SD is generally accepted to be the key determinants during the initiation event, several other sequences surrounding the start codon are proposed to influence (Stenström et al., 2001b and references therein). The non-random distribution of nucleotides among the 40 bases surrounding the initiation codon (Schneider et al., 1986, Stenström et al., 2001a and references therein), indicates that there might be more signals in the mRNA primary structure that constitute the ribosome binding site during initiation of translation (Dreyfus, 1988). A region located downstream of the start codon has been suggested to influence translation initiation by mRNA-rRNA complementary base pairing (Faxén et al., 1991, O'Connor et al., 1999 and references therein; Stenström et al., 2001a and references therein). However, mutational alterations both in mRNA and 16S rRNA failed to support such complementary binding (Firpo and Dahlberg, 1998, O'Connor et al., 1999). Still, this region influences translation initiation in a context dependent manner (Stenström et al., 2001a, Stenström et al., 2001b). A CA repeat sequence in the region downstream of the initiation codon in mRNA, with or without a leader, increases the level of expression (Martin-Farmer and Janssen, 1999). More than 30 naturally leaderless mRNAs have been found in Archaea, Bacteria and Eucarya (Wu and Janssen, 1996). The AUG initiation codon itself, not the codon – anti-codon contact is important for translation of leaderless mRNA in Escherichia coli (vanEtten and Janssen, 1998). The question is if the initiation triplet is the sole determinant for a proper initiation event when a beneficial upstream sequence is missing, or if there is a contribution also from the region downstream of the initiation codon. Since earlier reports have suggested a strong influence by the downstream region (DR) upon translation initiation or during the early elongation phase, we wanted to check gene expression associated with different naturally occurring DRs. The fourteen DRs that are analyzed in this report originate from genes that are naturally expressed at different levels, keeping in mind that they are under control of different promoters. We also analyzed context mutants in these DRs and how a stretch of lysine codons would influence expression. Our analysis shows that the base composition of the early codons, and not the amino acid sequence at the N-terminus of the gene protein product, is the determinant for gene expression.
Section snippets
Chemicals
Restriction enzymes and T4 DNA ligase were from Promega or BioLabs. DNA extraction kit was from Qiagen. Plasmids were prepared with a JET Prep purification system from Genomed GmbH. Termination mixtures for automatic sequencing were purchased from PerkinElmer.
Bacterial strain
The Δlac E. coli strain MC 1061 (Stenström et al., 2001b and references therein) was the host strain for all plasmids.
Plasmid and plasmid construction
Constructions of plasmids were done using standard recombinant DNA techniques. The Shine-Dalgarno region of pCMS71 (
The effect of natural DR sequences on gene expression
In this study two different Shine-Dalgarno (SD) sequences were used upstream of a lacZ reporter gene. The plasmid pCMS71 (Stenström et al., 2001b) contains a sequence designed to show low complementarity to the anti-SD region on the 16S rRNA (SD−; UAAAUAAA), while the derivative pCMS80 (Stenström et al., 2001a) contains a canonical Shine-Dalgarno sequence (SD+; UAAGGAGG). Both Shine-Dalgarno sequences were inserted with an appropriate spacing of eight nucleotides to the initiation codon (Fig. 1
A strong DR can act together with the SD to increase the translation initiation
Previous studies have shown that the downstream sequence located immediately 3′ of the initiation triplet affects gene expression (Faxén et al., 1991, O'Connor et al., 1999, Stenström et al., 2001b) at the level of translation initiation and/or early elongation (Stenström et al., 2001a). The results presented here are obtained by analysis of natural DRs composed of the five codons that follow the initiation codon and variants thereof. The effects by the DRs on gene expression are not correlated
Acknowledgements
This work has been supported by grants from NFR, SSF and TFR to LAI.
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