Expression of the Bacillus subtilis trpEDCFBA operon is influenced by translational coupling and Rho termination factor.

The trp RNA-binding attenuation protein (TRAP) regulates expression of the Bacillus subtilis trpEDCFBA operon by transcription attenuation and translational control mechanisms. Both mechanisms require binding of tryptophan-activated TRAP to 11 (G/U)AG repeats in the trp leader transcript. trpE translational control involves formation of a TRAP-dependent RNA structure that sequesters the trpE Shine-Dalgarno (SD) sequence (the SD blocking hairpin). By comparing expression levels from trpE'-'lacZ translational fusions controlled by the wild-type leader or by a leader that cannot form the SD blocking hairpin, we found that translational control requires a tryptophan concentration higher than that required for transcription attenuation. We also found that inhibition of trpE translation by the SD blocking hairpin does not alter the stability of the downstream message. Since the coding sequences for trpE and trpD overlap by 29 nucleotides, we examined expression levels from trpED'-'lacZ translational fusions to determine if these two genes are translationally coupled. We found that introduction of a UAA stop codon in trpE resulted in a substantial reduction in expression. Since expression was partially restored in the presence of a tRNA suppressor, our results indicate that trpE and trpD are translationally coupled. We determined that the coupling mechanism is TRAP independent and that formation of the SD blocking hairpin regulates trpD translation via translational coupling. We also constructed a rho mutation to investigate the role of Rho-dependent termination in trp operon expression. We found that TRAP-dependent formation of the SD blocking hairpin allows Rho access to the nascent transcript, causing transcriptional polarity.

Specificity determinants for the pyruvate dehydrogenase component reaction mapped with mutated and prosthetic group modified lipoyl domains.

Efficient catalysis in the second step of the pyruvate dehydrogenase (E1) component reaction requires a lipoyl group to be attached to a lipoyl domain that displays appropriately positioned specificity residues. As substrates, the human dihydrolipoyl acetyltransferase provides an N-terminal (L1) and an inner (L2) lipoyl domain. We evaluated the specificity requirements for the E1 reaction with 27 mutant L2 (including four substitutions for the lipoylated lysine, Lys(173)), with three analogs substituted for the lipoyl group on Lys(173), and with selected L1 mutants. Besides Lys(173) mutants, only E170Q mutation prevented lipoylation. Based on analysis of the structural stability of mutants by differential scanning calorimetry, alanine substitutions of residues with aromatic side chains in terminal regions outside the folded portion of the L2 domain significantly decreased the stability of mutant L2, suggesting specific interactions of these terminal regions with the folded domain. E1 reaction rates were markedly reduced by the following substitutions in the L2 domain (equivalent site-L1): L140A, S141A (S14A-L1), T143A, E162A, D172N, and E179A (E52A-L1). These mutants gave diverse changes in kinetic parameters. These residues are spread over >24 A on one side of the L2 structure, supporting extensive contact between E1 and L2 domain. Alignment of over 40 lipoyl domain sequences supports Ser(141), Thr(143), and Glu(179) serving as specificity residues for use by E1 from eukaryotic sources. Extensive interactions of the lipoyl-lysine prosthetic group within the active site are supported by the limited inhibition of E1 acetylation of native L2 by L2 domains altered either by mutation of Lys(173) or enzymatic addition of lipoate analogs to Lys(173). Thus, efficient use by mammalian E1 of cognate lipoyl domains derives from unique surface residues with critical interactions contributed by the universal lipoyl-lysine prosthetic group, key specificity residues, and some conserved residues, particularly Asp(172) adjacent to Lys(173).

trp RNA-binding attenuation protein-5' stem-loop RNA interaction is required for proper transcription attenuation control of the Bacillus subtilis trpEDCFBA operon.

The trp RNA-binding attenuation protein (TRAP) regulates expression of the Bacillus subtilis trpEDCFBA operon by a novel transcription attenuation mechanism. Tryptophan-activated TRAP binds to the nascent trp leader transcript by interacting with 11 (G/U)AG repeats, 6 of which are present in an antiterminator structure. TRAP binding to these repeats prevents formation of the antiterminator, thereby promoting formation of an overlapping intrinsic terminator. A third stem-loop structure that forms at the extreme 5' end of the trp leader transcript also plays a role in the transcription attenuation mechanism. The 5' stem-loop increases the affinity of TRAP for trp leader RNA. Results from RNA structure mapping experiments demonstrate that the 5' stem-loop consists of a 3-bp lower stem, a 5-by-2 asymmetric internal loop, a 6-bp upper stem, and a hexaloop at the apex of the structure. Footprinting results indicate that TRAP interacts with the 5' stem-loop and that this interaction differs depending on the number of downstream (G/U)AG repeats present in the transcript. Expression studies with trpE'-'lacZ translational fusions demonstrate that TRAP-5' stem-loop interaction is required for proper regulation of the trp operon. 3' RNA boundary experiments indicate that the 5' structure reduces the number of (G/U)AG repeats required for stable TRAP-trp leader RNA association. Thus, TRAP-5' stem-loop interaction may increase the likelihood that TRAP will bind to the (G/U)AG repeats in time to block antiterminator formation.

Effects of mutations in the L-tryptophan binding pocket of the Trp RNA-binding attenuation protein of Bacillus subtilis.

The Bacillus subtilis tryptophan biosynthetic genes are regulated by the trp RNA-binding attenuation protein (TRAP). Cooperative binding of L-tryptophan activates TRAP so that it can bind to RNA. The crystal structure revealed that L-tryptophan forms nine hydrogen bonds with various amino acid residues of TRAP. We performed site-directed mutagenesis to determine the importance of several of these hydrogen bonds in TRAP activation. We tested both alanine substitutions as well as substitutions more closely related to the natural amino acid at appropriate positions. Tryptophan binding mutations were identified in vivo having unchanged, reduced, or completely eliminated repression activity. Several of the in vivo defective TRAP mutants exhibited reduced affinity for tryptophan in vitro but did not interfere with RNA binding at saturating tryptophan concentrations. However, a 10-fold decrease in TRAP affinity for tryptophan led to an almost complete loss of regulation, whereas increased TRAP affinity for tryptophan had little or no effect on the in vivo regulatory activity of TRAP. One hydrogen bond was found to be dispensable for TRAP activity, whereas two others appear to be essential for TRAP function. Another mutant protein exhibited tryptophan-independent RNA binding activity. We also found that trp leader RNA increases the affinity of TRAP for tryptophan.

Requirements for the adaptor protein role of dihydrolipoyl acetyltransferase in the up-regulated function of the pyruvate dehydrogenase kinase and pyruvate dehydrogenase phosphatase.

The dihydrolipoyl acetyltransferase (E2 component) is a 60-mer assembled via its COOH-terminal domain with exterior E1-binding domain and two lipoyl domains (L2 then L1) sequentially connected by mobile linker regions. E2 facilitates markedly enhanced function of the pyruvate dehydrogenase kinase (PDK) and pyruvate dehydrogenase phosphatase (PDP). Human E2 structures were prepared with only one lipoyl domain (L1 or L2) or with alanines substituted at the sites of lipoylation (Lys-46 in L1 or Lys-173 in L2). The L2 domain and its lipoyl group were shown to be essential for markedly enhanced PDP function and were required for greatly up-regulated PDK function. The complete absence of the L1 domain reduced the enhancements of both of these activities but not the maximal effector-stimulated PDK activity through acetylation of L2. With nonlipoylated L2 present, lipoylated L1 supported a lesser enhancement in PDK function with significant stimulation upon acetylation of L1. Prevention of L1 lipoylation in K46AE2 removed this competitive L1 role and enhanced L2-facilitated PDK activity beyond that of native E2 when PDK activity was measured in the absence or in the presence of stimulatory effectors. Thus, the E2-L2 domain has a paramount role in facilitating enhanced PDK and PDP function but inclusion of E2-L1 domain, even in a noninteracting (nonlipoylated) form, contributes to the marked elevation of these activities.

Green fluorescent protein purification by organic extraction.

Green fluorescent protein (GFP) is widely used as an excellent reporter molecule in biochemistry and cell biology. Some biochemical and immunological assays require high-purity GFP. However, the majority of current procedures for GFP purification include multiple time-consuming chromatography steps with a low yield of the desired product or require tag-containing proteins. An alternative method is described for the GFP purification without affinity extensions using organic extraction yielding a highly homogeneous protein indistinguishable in spectroscopic properties from that purified by previous methods.

Protein kinase C in rod outer segments: effects of phosphorylation of the phosphodiesterase inhibitory subunit.

The inhibitory subunit (PDE gamma) of the cGMP phosphodiesterase (PDE alpha beta gamma 2) in rod outer segments (ROS) realizes its regulatory role in phototransduction by inhibition of PDE alpha beta catalytic activity. The photoreceptor G-protein, transducin, serves as a transducer from the receptor (rhodopsin) to the effector (PDE) and eliminates the inhibitory effect of PDE gamma by direct interaction with PDE gamma. Our previous study [Udovichenko, Cunnick, Gonzalez and Takemoto (1994) J: Biol. Chem. 269, 9850-9856] has shown that PDE gamma is a substrate for protein kinase C (PKC) from ROS and that phosphorylation by PKC increases the ability of PDE gamma to inhibit PDE alpha beta catalytic activity. Here we report that transducin is less effective in activation of PDE alpha beta (gamma p)2 (a complex of PDE alpha beta with phosphorylated PDE gamma, PDE gamma p) than PDE alpha beta gamma 2. PDE gamma p also increases the rate constant of GTP hydrolysis of transducin (from 0.16 S-1 for non-phosphorylated PDE gamma to 0.21 s-1 for PDE gamma p). These data suggest that phosphorylation of the inhibitory subunit of PDE by PKC may regulate the visual transduction cascade by decreasing the photoresponse.