A uridine-rich sequence required for translation of prokaryotic mRNA.

Abstract

Binding of 30S ribosomal subunits to mRNA during the initiation of prokaryotic translation is known to be influenced by the initiation codon and the Shine-Dalgarno sequence. Site-directed mutagenesis ofrnd, the Escherichia cofi gene encoding RNase D, has now shown that a Us sequence upstream of the Shine-Dalgarno region is also essential for expression of this mRNA. Alteration of two to five uridine residues within this sequence has no effect on mRNA levels but decreases RNase D protein and activity by as much as 95%, indicating that the U-rich sequence acts as an enhancer of translation. Moreover, mutant transcripts bind to 30S ribosomes in vitro with lower affinity than their wild-type counterparts, suggesting that the role of the Us sequence is in the initial binding of ribosomes to the translation initiation region of the message. These data demonstrate that sequences other than those previously recognized can be essential for translation initiation.Selection of the correct initiation codon by 30S ribosomal subunits is a primary determinant of accurate translation of mRNA in prokaryotic cells. However, despite extensive study, it is still not understood how this sequence of three nucleotides is distinguished from other residues in a message (1, 2). Factors known to influence the efficiency of binding of ribosomes to the translation initiation region of a mRNA include the initiation codon itself, a run of three to seven nucleotides upstream ofthe initiation codon termed the ShineDalgarno sequence, the spacing between these two segments, and the secondary structure of the initiation region (3,4). However, statistical analyses (5) and isolation of functional translation initiation regions (6) have shown that other sequences in these regions are nonrandom and may also play a role in the initiation process. Several possibilities for sequences upstream of the Shine-Dalgarno region that might function as translational enhancers have been proposed (7,8), although their significance is unclear. In addition to the RNA-RNA interactions between mRNA and ribosomes, it is likely that ribosomal proteins also participate in the selection of translation start sites, and protein S1 of the 30S subunit, in particular, has been implicated in this process (9). Based on UV crosslinking of protein S1 to phage and bacterial messages in vitro, it was suggested recently that pyrimidine-rich regions upstream of the Shine-Dalgarno sequence might interact with protein S1 and serve as ribosome recognition sites (10).During the course of studying the Escherichia coli rnd gene, which encodes the tRNA-processing enzyme RNase D, we identified (11, 12) a potential stem-loop structure followed by eight uridine residues located upstream of the initiator UUG codon and the Shine-Dalgarno sequence in the mRNA (Fig. 1). Although such a structure has features of a transcription terminator, when the stem-loop and uridine residues were deleted neither the rnd mRNA level nor the transcription start site was altered, but RNase D expression was el...

Design criteria for synthetic riboswitches acting on transcription

“…Both constructs differ in their overall activity, because in RS10-10-10, the ribosomal binding site is located immediately downstream of the riboswitch U-stretch that has a strong positive impact on translation efficiency. 12,27 Accordingly, gene expression is reduced in the shifted counterpart. In this tridem, a further increase of the activation ratio compared to the tandem is not possible, as the theophylline-independent activity is already close to zero in RS10-10shift.…”

“…Furthermore, as the close proximity of the terminator U stretch and the downstream Shine-Dalgarno sequence leads to a strong theophylline-independent activity of the reporter gene due to an enhanced translation rate (see ref. 27 ), we additionally designed serial RS constructs based on a modified riboswitch RS10shift. 12 In these constructs, a 19 nt insertion increases the distance between riboswitches and Shine-Dalgarno sequence, leading to a dramatic reduction of activity without theophylline (Fig.…”

An AUG initiation codon, not codon–anticodon complementarity, is required for the translation of unleadered mRNA in Escherichia coli

“…The RBS of conventionally leadered Escherichia coli mRNA extends approximately Ϯ 15 nucleotides relative to the start codon (Steitz, 1969;Steitz and Jakes, 1975;Steitz and Steege, 1977), contains non-random sequence using statistical analysis (Scherer et al, 1980;Stormo et al, 1982), and is responsible for establishing a 1000-fold range of translational efficiencies (Ray and Pearson, 1974;. Highly efficient initiation regions include some or all of the following mRNA elements: a polypyrimidine tract for ribosomal protein S1 interaction (Boni et al, 1990;Zhang and Deutscher, 1991;Tzareva et al, 1994); a Shine-Dalgarno (SD) sequence with basepairing complementarity to the anti-SD (ASD) of the 16S rRNA (Shine and Dalgarno, 1974;Hui and de Boer, 1987;Jacob et al, 1987); a cognate start codon for initiator fMet-tRNA anticodon interaction (Ringquist et al, 1992;Vollenoweth and Rabinowitz, 1992); and base-specific enhancer sequences upstream (Olins and Rangwala, 1989) or downstream (Sprengart et al, 1990; of the start codon. The relative importance and interdependence of these mRNA elements as well as the temporal order of their recognition by initiating ribosomes is less well understood.…”

Noncanonical repression of translation initiation through small RNA recruitment of the RNA chaperone Hfq

“…This behavior is explained by Hfq's preference to bind to AU-rich regions close to RBSs (Franze de Fernandez et al 1972;Senear and Steitz 1976), which are known to act as translational enhancers (Zhang and Deutscher 1992;Hook-Barnard et al 2007). In addition, Hfq is involved in RNA processing, as it regulates polyadenylation-dependent mRNA decay (Hajnsdorf and Regnier 2000;Mohanty et al 2004).…”