Synthesis of a bacteriophage MB78 late protein by novel ribosomal frameshifting

Abstract

MB78 is a virulent phage of Salmonella typhimurium that possesses a number of interesting features, making it a suitable organism to study the regulation of gene expression. A detailed physical map of this phage genome has been constructed and is being extensively studied at the molecular level. Here, we demonstrate the expression of two late proteins of bacteriophage MB78 derived from the same gene as a result of possible ribosomal frameshifting. In vitro transcription-translation yields a major protein that migrates as 28kDa, whereas in vivo expression using pET expression vectors yields two equally expressed proteins of molecular sizes 28 and 26kDa. A putative slippery sequence TTTAAAG and a pseudoknot structure, two essential cis elements required for the classical ribosomal frameshifting, are identified in the reading frame. Mutations created at the slippery sequence resulted in a single 28kDa protein and completely abolished the expression of 26kDa protein. Thus, we have produced the first evidence that ribosomal frameshifting occurs in bacteriophage MB78 of Salmonella typhimurium.

Influence of the stacking potential of the base 3??? of tandem shift codons on ???1 ribosomal frameshifting used for gene expression

“…The A AAA AAG motif, found in dnaX and in many insertion sequences of the IS3 family, is probably the most efficient heptameric Ϫ1 shift motif in E. coli+ But other heptamers are used for Ϫ1 programmed frameshifting, such as A AAA AAC in IS1 (Sekine & Ohtsubo, 1992), U UUA AAG in Salmonella typhimurium phage MB78 (Kolla et al+, 2000), and A AAA AAA in IS600 (M+F+ Prère & O+ Fayet, unpubl+)+ To determine if the 39 FIGURE 3. Incidence of reporters and stimulators+ A: The wild-type region (Fig+ 1) and the three mutants, in which the first nucleotide of SP2 is changed to A, C, or U, were cloned in the pGM-1, pOFX258, and pOFX317 vectors+ Frameshifting frequency was measured by pulse-labeling on four clones for each construct (the 95% confidence interval of the mean is indicated)+ B: The frameshift regions of IS222 (Kropinski et al+, 1994) and dnaX (Larsen et al+, 1997) were cloned into pOFX302+ Frameshifting frequencies were estimated by b-galactosidase assay; note that for the IS911 constructs this method gives values three times lower than pulse labeling+ context effect was a general property of heptameric X XXY YYZ motifs, we created mutants of the IS911 region in which 17 other motifs were combined with each of the four possible nucleotides at the first position of SP2+ In Table 3, the b-galactosidase activity of these 17 motifs, plus A AAA AAG, is presented according to their 39 nucleotide+ The 18 heptamers tested can be separated into two classes according to the 39 context effect+ There is one special case, construct A AAA AAA A, not discussed further, because its high level of activity is due to transcriptional slippage (data not shown) rather than frameshifting+ Motifs of the first class, the four X XXA AAG heptamers, display a 39 context effect identical, in pattern and magnitude, with that initially found with A AAA AAG+ They give a relatively high level of frameshifting (from 98 to 425 b-galactosidase units), except U UUA AAG (for reasons yet unclear, U UUA restricts frameshifting); the four of them are not efficient at all in eukaryotes (Brierley et al+, 1992)+ The common element of these motifs, the A AAG tetramer, probably fixes the pattern of the 39 context effect+…”

Temperature Sensitive Mutation in the 38 kDa Minor Structural Protein Gene of Phage MB78 Interferes with Phage Morphogenesis

“…Two strong promoters of the phage have been studied in details [5,6]. A number of restriction fragments of the phage genome have been cloned in M13mp 11 and some of the fragments have been studied extensively [6][7][8][9][10][11]. MB78 does not seem to be related to other phages since the ORFs so far identified do not show any similarity with reported phage proteins.…”