Since the discovery that prokaryotic Sec insertion is dependent on a downstream mRNA sequence (14
) that is recognized by SelB (24
), several groups have attempted to define the recognition elements within the SECIS, to further elucidate the mechanism of Sec insertion and to minimize the sequence constraints. In the first work to substantially minimize the SECIS requirements relative to the wt sequence, Engelberg-Kulka and coworkers (11
) investigated a variety of SECIS variants cloned upstream of β-galactosidase and measured readthrough and radioactive selenium incorporation. A critical determination was that the lower stem (positions +4 to +14 and +32 to +41) could be completely unpaired with no deleterious effect on opal suppression. A series of upper stem variants was prepared, in the context of a fully unpaired lower stem, and it was concluded that the upper stem must be fully paired.
Using the same selenopeptide–pIII fusion vector as in the present work, our earlier phage display studies (16
) confirmed that the lower stem nucleotides in positions +4 to +12 could be randomized, in the context of the wt upper stem and loop, with efficient Sec insertion. The randomization scheme in both studies was (NNK) for each codon downstream of UGA, where N is any nucleotide and K is G or T. Fortuitously, none of the positions randomized as K were G or T in the wt paired lower stem, so our SECIS variants were inherently biased against a paired lower stem. Moreover, in the present study we randomized the lower stem nucleotides +13, +14, +32 and +33 and obtained functional SECIS variants with almost every possible combination of nucleotides in these positions (data not shown). Our data from screening combinatorial libraries of lower stem sequences demonstrates that the lower stem can indeed be fully unpaired, and that this region is tolerant to highly randomized sequences.
The goal of the present work was to employ combinatorial methods to determine the requirements of the upper stem and loop regions of the SECIS. Previous studies of the upper stem and loop (11
) investigated individual SECIS variants, rather than performing a thorough sampling of sequence space. In one study (25
), a combinatorial aptamer approach was employed to identify upper stem–loop sequences that bound SelB in vitro
. Although that work did underscore the importance of the upper stem bulged uridine in SelB recognition, the aptamers were screened in vitro
for SelB binding, and many SelB binding sequences were not functional in vivo
for Sec insertion. Our goal was to perform library screening in vivo
(i.e. during phage morphogenesis) in order to identify functional SECIS variants.
Our findings revealed an unexpected degree of flexibility in the SECIS requirements (Fig. ). The upper stem was considerably more tolerant to mutations than predicted. The positions closest to the loop were somewhat fixed, with only two functional variants (SV6 and SV13) arising in the pair closest to the loop (+19 and +28) and three (SV9, SV13 and SV19) in the next pair down (+18 and +29). In contrast the lower two pairs in the upper stem (+15 and +31, and +16 and +30) had a wide range of permissible mutations. SV2 was especially relevant, because its G15C mutation had been shown previously to abolish UGA readthrough in the context of a completely unpaired lower stem (11
). To further investigate the interaction between a non-native upper stem and the pairing of the lower stem, SECIS variants SV8 and SV15 were prepared with a mostly unpaired lower stem. The resulting clones, SV8* and SV15*, permitted selenium-dependent UGA readthrough, but less than half as much as their parent clones (Fig. ). Similar results were observed using a SECIS variant selected from RNA aptamers for its SelB-binding ability (25
): a clone with a non-native upper stem revealed no readthrough with a non-native lower stem, but suppression rose to 17% when the upper stem variant was fused to the wt lower stem. These results together suggest that the SECIS requirements are not limited to the upper stem. Perhaps some minimal level of stem pairing is required in order to present the required elements for SelB recognition, and this pairing can be distributed over the entire length of the stem, rather than just the upper region.
Although Heider et al
) observed up to 22% readthrough with certain loop mutations, we did not observe any permissible loop variants using our randomization scheme. The loop results might have been different in the context of a completely wt stem; the combined effects of upper stem and loop mutations presumably diminished the chances of identifying functional loop variants. In two cases (SV15 and SV17), the ‘required bulged’ U17 was mutated to a G; in both of these cases, however, G16 was mutated to a U, and U18 was unmutated, so that there were two uridines, either of which could possibly function as the recognition element for SelB. Further experiments are necessary to fully delineate the importance of the bulged U in the upper stem. For many of the SECIS variants studied, molecular modeling of the RNA sequences did not portray the bulged uridine (data not shown), but it is likely that the protein–RNA contacts afforded by SelB binding would stabilize the appropriate binding conformation.
Although much of the Sec insertion mechanism has been defined, the nature of the SelB–SECIS recognition is not fully elucidated. It has been shown by binding and toeprinting studies that the upper stem and loop region of the SECIS is contacted by SelB (24
). In the absence of structural data showing the SelB–SECIS complex, it is difficult to predict the exact recognition elements in the SECIS. Given the permissiveness of the entire stem, it seems likely that SelB principally recognizes the loop and bulged U, but probably does not recognize sequence or structural elements within the stem. The stem probably does, however, need to be stable enough to present the appropriate bulged U and loop for SelB recognition.
Based on our earlier results (17
), we expected the TGAGwt SECIS sequence to allow Trp-inserting opal suppression in addition to Sec insertion. Although the β-galactosidase assay did reveal a somewhat elevated level of readthrough in the absence of supplemental selenite, and overall readthrough higher than TGACwt SECIS, this clone still had substantial selenium enhancement of both lacZα readthrough and phage plaque diameter. This result is consistent with the report that the Arg and Val codons immediately upstream of Sec in the native fdh sequence, together with a TGAC sequence, help to prevent readthrough when selenium concentration is low (13
). In our previous phage display study we randomized the four codons upstream of Sec, and found the resulting TGAG clones to have selenium-independent UGA readthrough as measured by phage production and plaque diameter. In the present work the upstream codons were fixed as the native sequence (Arg-Val), which partially counteracted the tendency of the downstream G to direct Trp-inserting opal suppression in the absence of selenium.
Overexpression of the SelABC genes has been shown to enhance selenoprotein expression in the context of a native SECIS (5
). Our early attempts at screening for functional SECIS variants were performed in E.coli
ER2738, without the pSelABC plasmid. These efforts were hampered by the instability of many Sec-inserting clones; phage carrying mutations in the UGA codon had a significant growth advantage over UGA-containing clones. The overexpression of the SelABC genes stabilized the clones during amplification so that we could identify SECIS variants. To ascertain that these variants were functional in the normal E.coli
background, the β-galactosidase assays shown in Figures and were performed in ER2738 without pSelABC. It is expected that overexpression of the SelABC genes would enhance selenoprotein expression in the context of any of our SECIS variants. This would be especially useful if protein sequence requirements dictated the use of a minimally efficient SECIS variant.
In addition to the growing number of approaches for obtaining selenoproteins [e.g. expression in cultured Chlamydomonas reinhardtii
plant cells (30
)], expression in E.coli
may be a viable option for more selenoprotein sequences than previously predicted. Although our initial attempts revealed no permissible variations in the SECIS loop sequence, the upper stem requirements appear to be substantially more flexible than had been reported. It seems plausible that the ‘rules’ of SECIS structure are not as easily defined as thought previously: while the full-length SECIS requires some minimum level of hairpin stability, this stability can evidently be distributed along the length of the stem structure. It may be possible to express selenoproteins in E.coli
by simply overexpressing the SelABC genes and designing a SECIS sequence that allows expression of the native downstream amino acid sequence of the protein, but which preserves the base-pairing and length of the stem region and as much of the sequence of the loop region as possible.