The Highly Efficient Translation Initiation Region from the Escherichia coli rpsA Gene Lacks a Shine-Dalgarno Element

Abstract

The translational initiation region (TIR) of the Escherichia coli rpsA gene, which encodes ribosomal protein S1, shows a number of unusual features. It extends far upstream (to position ؊91) of the initiator AUG, it lacks a canonical Shine-Dalgarno sequence (SD) element, and it can fold into three successive hairpins (I, II, and III) that are essential for high translational activity. Two conserved GGA trinucleotides, present in the loops of hairpins I and II, have been proposed to form a discontinuous SD. Here, we have tested this hypothesis with the "specialized ribosome" approach. Depending upon the constructs used, translation initiation was decreased three-to sevenfold upon changing the conserved GGA to CCU. However, although chemical probing showed that the mutated trinucleotides were accessible, no restoration was observed when the ribosome anti-SD was symmetrically changed from CCUCC to GGAGG. When the same change was introduced in the SD from a conventional TIR as a control, activity was stimulated. This result suggests that the GGA trinucleotides do not form a discontinuous SD. Others hypotheses that may account for their role are discussed. Curiously, we also find that, when expressed at moderate level (30 to 40% of total ribosomes), specialized ribosomes are only twofold disadvantaged over normal ribosomes for the translation of bulk cellular mRNAs. These findings suggest that, under these conditions, the SD-anti-SD interaction plays a significant but not essential role for the synthesis of bulk cellular proteins.

Regulation of the rplY gene encoding 5S rRNA binding protein L25 in Escherichia coli and related bacteria

“…3C). This is reminiscent of the fis (Nafissi et al 2012) and rpsA (Boni et al 2000(Boni et al , 2001Skorski et al 2006) mRNAs that lack true SD-elements; nevertheless, they are translated very efficiently due to the presence of the AU-rich translational enhancers and specific secondary structures within their FIGURE 2. Regulation of the rplY expression at the translation level.…”

“…In E. coli, mRNAs capable of providing a high level of expression in the absence of a canonical SD element are rare: As yet, only rpsA (Boni et al 2001;Skorski et al 2006), fis (Nafissi et al 2012), and rplY (this work) mRNAs can be attributed to this category. The replacement of the rplY GAGAG SD-like sequence by a classic GGAGG-element abolished L25-mediated control and reduced rather than increased the translation yield.…”

Proximity of the Start Codon to a Leaderless mRNA's 5′ Terminus Is a Strong Positive Determinant of Ribosome Binding and Expression in Escherichia coli

“…Although a Shine-Dalgarno (SD) sequence (25) is known to speed the formation of initiation complexes and result in higher expression, prokaryotes also contain noncanonical mRNAs, many of which lack a SD-type sequence but are highly expressed (6,9,24,26). Leaderless mRNAs carrying a start codon at their 5Ј terminus are a group of noncanonical mRNAs found in all domains of life (5,8,15,23).…”

Robust Translation of the Nucleoid Protein Fis Requires a Remote Upstream AU Element and Is Enhanced by RNA Secondary Structure

“…Nevertheless, there are a limited number of examples of upstream secondary structures that function to enhance translation. The best-studied examples are the stem-loop structures increasing translation of rnd, rpsA, and csgD, but their mechanisms of action remain poorly understood (5,20,50,60). The prominent effects of RNA sequences that are likely to form secondary structures upstream of the AU element in fis raise the possibility of riboswitch-like mechanisms or binding sites for trans-acting small untranslated RNA or protein factors influencing fis translation.…”