Genetic analysis of bacteriophage lambda cIII gene: mRNA structural requirements for translation initiation

Abstract

The bacteriophage A cIl gene product regulates the lysogenic pathway. The cIl gene is located in the leftward operon, which is transcribed from the PL promoter. We have previously shown (S. Altuvia and A. B. Oppenheim, J. Bacteriol. 167:415-419, 1986) that mutations that show elevated expression lie within the clll coding sequence. We isolated mutants that show decreased CIII activity. AUl the mutations were found to cause a drastic reduction in the rate of initiation of cHl translation. Several mutations were found to be scattered within the first 40 nucleotides of the cIl coding region. Additional mutations affected the AUG initiation codon, the Shine-Dalgarno sequence, and the upstream RNaseIII processing site. Computer folding of the cIl mRNA suggested the presence of two alternative RNA structures. All the mutations within the coding region that reduce expression reduce the stability of one specific mRNA structure (structure B). Mutations that increase expression lie in the loops of this structure and may in fact stabilize it by interfering with the formation of the alternative structure (structure A). Thus, it appears that a specific mRNA secondary structure at the beginning of the cIl coding region is essential for efficient translation, suggesting that changes in mRNA structure regulate cm expression.Initiation of translation in procaryotes has been intensively investigated (for recent reviews, see references 14 and 42). Some structural elements important for mRNA recognition by the ribosome have been defined, namely, the Shine-Dalgarno sequence (39), the initiation codon, the spacing between these two elements, and other nonrandomly occurring nucleotides before and after the initiation codon (e.g., see reference 20).Many experiments also suggested that, to be available for ribosome recognition, the ribosome-binding region must be free of occluding secondary structures that may block ribosome access; i.e., it must either be located in a region free of secondary structures or be located in a hairpin loop (14,17,22,46). There are examples in which opening of such an joccluding secondary structure by incoming ribosomes is used to achieve translational coupling between bacteriophage genes (6, 8). In other cases, alternative mRNA structures have been proposed as a way to control expression of drug resistance genes (11,31,33).Infection of Escherichia coli by bacteriophage X may lead to a lytic or a lysogenic response (18). In the lysogenic response, cll and cIII gene products play a critical role. The cll gene product acts as a positive regulator and activates several phage promoters (12,47). It is a highly unstable protein with a half-life of 1.5 min (16). The clll gene product, a 54-amino-acid-long protein (25), was found to stabilize the CII protein in vivo, extending its half-life (19,34). It was also found to induce the heat shock response, probably through stabilization of C&32 (3). Phage mutants defective in the clll gene lysogenize poorly (24), whereas elevated expression of CIII caused by point mutat...

The HflB protease of Escherichia coli degrades its inhibitor lambda cIII

“…Plasmid pSTD61 (3) carries the ATPasenegative hflB gene cloned downstream of the lac promoter in pHSG575. Plasmid ptaccIII (27) carries the CIII gene under control of the IPTG (isopropyl-␤-Dthiogalactopyranoside)-inducible tac promoter of the ampicillin-resistant vector ptac 12H. Plasmid pCG179 (42) carries the rpoH gene under control of the tac promoter.…”

“…cIII is itself unstable (27), implying the existence of a proteolytic system capable of degrading it. The known properties of cIII-stabilization of cII and 32 and growth inhibition of the host cell-could be explained if cIII inhibits or competes efficiently for HflB.…”

Genetic analysis of the cIII gene of bacteriophage HK022

“…Previous experiments have shown that the host protein RNase III is required for efficient cIIl translation (2). In addition, genetic and biochemical evidence demonstrated that the region around the clII ribosome-binding site is found in two alternative conformations, only one of which is translated (1,16). We suggested that these features are related to regulation of clll expression at the translational level.…”

“…In HK022, however, in contrast to X, no additional cleavage sites could be detected within the coding region (3a; data not shown). (1,2,16) in spite of extensive sequence dissimilarities between the HK022 and A clII coding regions (18) (Fig. 7).…”

“…Plasmid ptacXclll, carrying the X cIll gene under tac promoter control, has been described previously (16). The plasmid used here is a mutant plasmid, obtained by site-directed mutagenesis, which yields a nonfunctional CIII protein (16a).…”