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

The bacteriophage lambda cIII gene product regulates the lysogenic pathway. The cIII 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 cIII coding sequence. We isolated mutants that show decreased CIII activity. All the mutations were found to cause a drastic reduction in the rate of initiation of cIII translation. Several mutations were found to be scattered within the first 40 nucleotides of the cIII coding region. Additional mutations affected the AUG initiation codon, the Shine-Dalgarno sequence, and the upstream RNaseIII processing site. Computer folding of the cIII 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 cIII coding region is essential for efficient translation, suggesting that changes in mRNA structure regulate cIII expression.     

The HflB protease of Escherichia coli degrades its inhibitor lambda cIII.

The cIII protein of bacteriophage lambda is known to protect two regulatory proteins from degradation by the essential Escherichia coli protease HflB (also known as FtsH), viz., the lambda cII protein and the host heat shock sigma factor sigma32. lambda cIII, itself an unstable protein, is partially stabilized when the HflB concentration is decreased, and its half-life is decreased when HflB is overproduced, strongly suggesting that it is degraded by HflB in vivo. The in vivo degradation of lambda cIII (unlike that of sigma32) does not require the molecular chaperone DnaK. Furthermore, the half-life of lambda cIII is not affected by depletion of the endogenous ATP pool, suggesting that lambda cIII degradation is ATP independent (unlike that of lambda cII and sigma32). The lambda cIII protein, which is predicted to contain a 22-amino-acid amphipathic helix, is associated with the membrane, and nonlethal overproduction of lambda cIII makes cells hypersensitive to the detergent sodium dodecyl sulfate. This could reflect a direct lambda cIII-membrane interaction or an indirect association via the membrane-bound HflB protein, which is known to be involved in the assembly of certain periplasmic and outer membrane proteins.     

Alternative mRNA structures of the cIII gene of bacteriophage lambda determine the rate of its translation initiation

The bacteriophage lambda cIII gene product has a regulatory function in the lysis-lysogeny decision following infection. The availability of a set of cIII expression mutants allowed us to establish the structure-function relationship of the cIII mRNA. We demonstrate, using defined in vitro systems, that the cIII mRNA is present in two conformations at equilibrium. Mutations that have been shown to lead to cIII overexpression were found to freeze the RNA in one conformation (structure B), and permit efficient binding to the 30 S ribosomal subunit. Mutations that have been shown to prevent cIII translation cause the mRNA to assume the alternative conformation (structure A). In this structure, the translation initiation region is occluded, thereby preventing 30 S ribosomal subunit binding. By varying the temperature or Mg2+ concentration it was possible to alter the relative proportion of the alternative structures in wild-type mRNA. We suggest that the regulation of the equilibrium between the two mRNA conformations provides a mechanism for the control of cIII gene expression.     

Insertion sequence IS2 associated with int-constitutive mutants of bacteriophage lambda.

We have examined mutations in bacteriophage lambda called int-c, which confer elevated constitutive expression on the int gene for prophage integration. One class of mutations, which map between the b538 and bio386 endpoints, does not appear to be associated with any major chromosomal modification, whereas the second class has the IS2 insertion sequence in orientation II within the region between gene int and the b538 endpoint, All int-c mutations are within gene xis, with the possible exception of int-c548, which might be located between int and xis. The present data are most consistent with the following notion: (1) the point mutations of class one inactivate the tI terminator signal of the pI-tI leader RNA for gene int and thus render int expression independent of the antiterminating action of the cII and cIII products, and (2) the second class of int-c mutants is constitutive for Int because the IS2 insertion, when strategically located between int and tI, provides a new constitutive promoter for int transciption.     

Bacteriophage lambda mutants (lambdatp) that overproduce repressor.

Lambda tp mutants, selected for their ability to form turbid plaques on lon hosts, overproduce repressor. The tp1 and tp2 mutations have been located within (or adjacent to) the cIII gene. The tp1 mutation reduced late gene expression, as measured by endolysin synthesis (in the absence of functional cI repressor) and progeny phage yield. The tp4 mutation was mapped in the cY-cII region, and complementation tests indicated that tp4 affects the diffusible product of the cII gene. The tp4 mutation also reduced progeny production, but did not markedly affect endolysin synthesis.     

Mitochondrial complex III Rieske Fe-S protein processing and assembly

Regulation of the mitochondrial respiratory chain biogenesis is a matter of great interest because of its implications for mitochondrial disease. One of the mitochondrial disease genes recently discovered associated to encephalopathy and mitochondrial complex III (cIII) deficiency is TTC19. Our study of TTC19-deficient human and mouse models, has led us to propose a post-assembly quality control role or ‘husbandry’ function for this factor that is linked to Rieske Fe-S protein (UQCRFS1). UQCRFS1 is the last incorporated cIII subunit, and its presence is essential for enzymatic activity. During UQCRFS1 assembly, the precursor is cleaved and its N-terminal part remains bound to the complex, between the two core subunits (UQCRC1 and UQCRC2). In the absence of TTC19 there is a prominent accumulation of these UQCRFS1-derived N-terminal fragments that proved to be detrimental for cIII function. In this article we will discuss some ideas around the UQCRFS1 processing and assembly and its importance for the regulation of cIII activity and biogenesis.     

Spontaneous lambda OR mutations suppress inhibition of bacteriophage growth by nonimmune exclusion phenotype of defective lambda prophage.

Survivor clones with defects in gene functions that participate in the replicative killing of thermally induced Escherichia coli constructs with integrated lambda N through P or cIII through P gene fragments were selected at a frequency of about 10(-6). Among the population of survivors, clones were identified that exhibited normal lambda immunity at 30 degrees C, as shown by their ability to prevent the plating of lambda wild type and to support the plating of a nearly identical heteroimmune bacteriophage lambda imm434. However, when placed at 42 degrees C to inactivate the cIts857 repressor, these survivor isolates excluded the plating of both lambda wild-type and lambda imm434 phages, a phenotype designated nonimmune exclusion (Nie). Spontaneous mutants of lambda wild type were isolated that overcame the Nie phenotype and would plaque at 42 degrees C on cell lawns of these isolates. The acquired lambda se mutations suppressed nonimmune exclusion, prevented lysogenization by interrupting repressor expression from PRM, and made the phage insensitive to replicative inhibition. The se mutations were genetically mapped and sequenced within the rightward lambda operator site.     

Chloramphenicol stimulation of lysogeny by lambda regulatory mutants.

Inhibition of protein synthesis in Escherichia coli by amino acid starvation or chloramphenicol addition increases the frequency of lysogeny by lambda phage two- to fourfold. Lambda cIII mutants, which normally lysogenize at very low frequencies, lysogenize at very high frequencies in the presence of chloramphenicol.     

The cIII gene and protein of bacteriophage lambda

The cIII and cII gene products of bacteriophage λ control the lysogenic response through positive regulation of the viral repressor and integration genes and negative regulation of lytic functions. Although many aspects of cII action have been defined biochemically, little is known about cIII. As a first step in defining the molecular role of cIII in the regulation of lysogeny, we have determined the precise location and DNA sequence of the cIII gene. In addition, we have identified the cIII gene product as a polypeptide with a molecular weight of approximately 6000.     

Stimulation of groE synthesis in Escherichia coli by bacteriophage lambda infection

We found that infection of Escherichia cell by lambda results in at least a twofold stimulation in the rate of synthesis of one of the products of groE. To determine what lambda-coded factors were responsible for this stimulation, numerous phage lambda mutants carrying bio substitutions were analyzed for their ability to stimulate groE synthesis. Our results revealed that the main factor(s) which is responsible for stimulating groE synthesis is located between the endpoints of the lambda bio69 and lambda bio252 substitutions, a region of DNA coding for bet, gam, kil, and cIII.     

Bioenergetic consequences from xenotopic expression of a tunicate AOX in mouse mitochondria: Switch from RET and ROS to FET

Electron transfer from all respiratory chain dehydrogenases of the electron transport chain (ETC) converges at the level of the quinone (Q) pool. The Q redox state is thus a function of electron input (reduction) and output (oxidation) and closely reflects the mitochondrial respiratory state. Disruption of electron flux at the level of the cytochrome bc₁ complex (cIII) or cytochrome c oxidase (cIV) shifts the Q redox poise to a more reduced state which is generally sensed as respiratory stress. To cope with respiratory stress, many species, but not insects and vertebrates, express alternative oxidase (AOX) which acts as an electron sink for reduced Q and by-passes cIII and cIV. Here, we used Ciona intestinalis AOX xenotopically expressed in mouse mitochondria to study how respiratory states impact the Q poise and how AOX may be used to restore respiration. Particularly interesting is our finding that electron input through succinate dehydrogenase (cII), but not NADH:ubiquinone oxidoreductase (cI), reduces the Q pool almost entirely (>90%) irrespective of the respiratory state. AOX enhances the forward electron transport (FET) from cII thereby decreasing reverse electron transport (RET) and ROS specifically when non-phosphorylating. AOX is not engaged with cI substrates, however, unless a respiratory inhibitor is added. This sheds new light on Q poise signaling, the biological role of cII which enigmatically is the only ETC complex absent from respiratory supercomplexes but yet participates in the tricarboxylic acid (TCA) cycle. Finally, we delineate potential risks and benefits arising from therapeutic AOX transfer.