Base content versus position and prevalence of cytosine after third round of selection.

<p>(A) Base content versus position for non-SD, SD, and all sequences is shown. In the non-SD group, the cytosine content is high at all positions. In the SD group, a high frequency of guanine is detected approximately between positions −12 and −9. (B) Histograms of cytosine counts in the randomized region for non-SD, SD, and all sequences are shown.</p

Distribution of potential sites for base-pairing of C-rich RBSs to 16S rRNA.

<p>Regions on the <i>E. coli</i> 30S ribosomal subunit with significant complementarity to the 30-5-3 library (<i>p</i>-value<0.01; Bonferroni-corrected) were determined. Significant seven-base windows that shared six bases with at least one neighboring significant window are highlighted in red (PyMOL rendering of PDB 3DF1). Four different views are shown to convey the general distribution of these potential base-pairing sites over the small ribosomal subunit. The first view shows the face that becomes buried after assembly with the large ribosomal subunit. The yellow ellipse indicates the approximate position of the anti-SD sequence. 16S rRNA = light gray; ribosomal proteins = dark gray.</p

Model for RBS functioning <i>in vitro</i> and <i>in vivo</i>.

<p>Of all possible RBSs, a certain subset works efficiently in a minimal, <i>E. coli-</i>based system. Of these, some RBSs work efficiently in <i>E. coli</i> (e.g., WT pRDV RBS), in other bacteria, and in distantly-related organisms, such as human, which contains many C-rich motifs near the start codon. It is likely that these three groups have some overlap (represented by dashed lines), but for the purposes of making generalizations, they have been drawn separately. Finally, certain RBSs that work efficiently in <i>E. coli</i> most likely require <i>in vivo</i> factors not present in the minimal system to function efficiently; the same can be said of certain RBSs that work efficiently in other bacteria or in human. Moreover, changing the context of an RBS may greatly change its efficiency and move it into a different space in the diagram. Nevertheless, broad-specificity mRNA-rRNA base-pairing suggested by our study using a minimal <i>E. coli</i>-based system may serve as a unifying mechanism for the functioning of a subset of RBSs from diverse hosts.</p

<i>In vitro</i> competition and <i>in vivo</i> expression.

<p>(A) WT and Clone 30-30-1-1 high C 1 were differentially affected by 400 µM 18-base ssDNA oligonucleotide competitors: random (N), clone 30-30-1-1 high C 1, a similar C-rich clone (30-5-3 high C 7), WT, and poly-C. MBP = maltose-binding protein. (B) Expression cassettes containing an RBS followed by FLAG-off7-emGFP were built by assembly PCR and cloned into pET-3a, which was used to transform BL21(DE3)pLysS. (C) Green fluorescence (excitation/emission: 487/509 nm) was quantified by flow cytometry after 4 h induction with 1 mM IPTG. The average median fluorescence of induced and uninduced clones is shown. Error bars represent standard deviation of at least three experiments. The first five sequences are the <i>E. coli</i> 5′ UTRs (18 bases before the start codon) having the most similarity to individual selected library members. They also happen to be highly C-rich for <i>E. coli</i>. Of these, only the <i>E. coli nrdB</i> 5′ UTR (UCCCAAC<u>AGGA</u>CACACUC) contains an SD motif (underlined). The next 15 sequences (“Clone 1” to “Clone 15”) are the most prevalent clones sequenced from the 30-5-3 selection scheme. The next six sequences are three of the most C-rich clones sequenced and three of the most C-rich 5′ UTRs present in phages from the EMBL-EBI database (<i>Burkholderia</i> phage KS14: HM461982; <i>Mycobacterium</i> phage Nigel: EU770221; and <i>Synechococcus</i> phage Syn5: EF372997, respectively). The final sequence is poly-C, which does not perform well. WT average median fluorescence (not shown) was extremely high (1417±178 AU induced, 15.2±15.6 AU uninduced).</p

Ribosome display, library context, and selection scheme.

<p>(A) Our adaptation of ribosome display for selection of efficiently translated sequences is shown. The naïve DNA library contained an 18-bp randomized RBS region prior to the start codon. Selection was performed by limiting the time of <i>in vitro</i> translation. Multiple rounds of increasing stringency were performed. (B) The context of the randomized RBS region is shown. Upstream is the T7 promoter and 5′ UTR of the ribosome display vector, pRDV, which is partially derived from phage. This region contains 89 bases in the DNA construct and 63 bases in the mRNA transcript (5′ UTR only). Downstream is a fusion protein consisting of a FLAG tag, off7 (a designed ankyrin repeat protein which binds maltose-binding protein), and tolA (an unstructured spacer derived from <i>E. coli</i> tolA which allows off7 to exit the ribosomal tunnel and fold properly). There is no stop codon. (C) The basic selection scheme is shown. The naïve RBS library was subjected to three selection rounds of increasing stringency: 30 min, 5 min, and 3 min translation. SD sequences were moderately enriched between rounds, but many non-SD sequences remained in the pool after the highly stringent 3 min translation.</p

Evidence for Context-Dependent Complementarity of Non-Shine-Dalgarno Ribosome Binding Sites to <i>Escherichia coli</i> rRNA

While the ribosome has evolved to function in complex intracellular environments, these contexts do not easily allow for the study of its inherent capabilities. We have used a synthetic, well-defined <i>Escherichia coli</i> (<i>E. coli</i>)-based translation system in conjunction with ribosome display, a powerful <i>in vitro</i> selection method, to identify ribosome binding sites (RBSs) that can promote the efficient translation of messenger RNAs (mRNAs) with a leader length representative of natural <i>E. coli</i> mRNAs. In previous work, we used a longer leader sequence and unexpectedly recovered highly efficient cytosine-rich sequences with complementarity to the 16S ribosomal RNA (rRNA) and similarity to eukaryotic RBSs. In the current study, Shine-Dalgarno (SD) sequences were prevalent, but non-SD sequences were also heavily enriched and were dominated by novel guanine- and uracil-rich motifs that showed statistically significant complementarity to the 16S rRNA. Additionally, only SD motifs exhibited position-dependent decreases in sequence entropy, indicating that non-SD motifs likely operate by increasing the local concentration of ribosomes in the vicinity of the start codon, rather than by a position-dependent mechanism. These results further support the putative generality of mRNA-rRNA complementarity in facilitating mRNA translation but also suggest that context (e.g., leader length and composition) dictates the specific subset of possible RBSs that are used for efficient translation of a given transcript