Role of the DNA sequence downstream of the Bacillus subtilis hut promoter in regulation of the hut operon

To identify the role of the downstream region of a hut promoter in regulation of the Bacillus subtilis hut operon, three single-base substitutions (+9G-->A, +14C-->T, and +23T-->G) were introduced into the hut operon. Analysis of expression of the hut operon containing each of these three single-base substitutions and the hut-lacZ fusions with the single-base substitutions at position +14 showed that the position at +14 and probably the position at +23 were required for amino acid repression at the hut promoter, while the position at +14 was not required for catabolite repression at the hut promoter. The position at +9 was required for a histidine-dependent increase of activity of the hut promoter. Analysis of expression of the hut-lacZ fusions and the hut operon in the codY mutant indicated that the position at +14 and probably the position at +23 were involved in CodY-mediated amino acid repression at the hut promoter and that CodY was not required for catabolite repression at the hut promoter.     

Lipoprotein sorting signals evaluated as the LolA-dependent release of lipoproteins from the cytoplasmic membrane of Escherichia coli

Escherichia coli lipoproteins are anchored to either the inner or outer membrane through fatty acyl chains covalently attached to an N-terminal cysteine. Aspartate at position 2 functions to retain lipoproteins in the inner membrane, although the retention is perturbed depending on the residue at position 3. We previously revealed that LolCDE and LolA play critical roles in this lipoprotein sorting. To clarify the sorting signals, the LolA-dependent release of lipoprotein derivatives having various residues at positions 2 and 3 was examined in spheroplasts. When the residue at position 3 was serine, only aspartate at position 2 caused the retention of lipoproteins in spheroplasts. We then examined the release of derivatives having aspartate at position 2 and various residues at position 3. Strong inner membrane retention occurred with a limited number of species of residues at position 3. These residues were present at position 3 of native lipoproteins having aspartate at position 2, whereas residues that inhibited the retention were not. It was also found that a strong inner membrane retention signal having residues other than aspartate at position 2 could be formed through the combination of the residues at positions 2 and 3. These results indicate that the inner membrane localization of native lipoproteins is ensured by the use of a limited number of strong inner membrane retention signals.     

Synthetic model proteins: the relative contribution of leucine residues at the nonequivalent positions of the 3-4 hydrophobic repeat to the stability of the two-stranded alpha-helical coiled-coil.

Our de novo designed coiled-coil model protein consists of two identical 35-residue polypeptide chains arranged in a parallel and in-register alignment via interchain hydrophobic interactions and a disulfide bridge at the position 2 between two helices. To quantitate the relative contribution of leucine residues at the nonequivalent position of the 3-4 hydrophobic repeat to the stability of the two-stranded alpha-helical coiled-coil, a single alanine was systematically substituted for a leucine in each chain at position "a" (9, 16, 23, or 30) or "d" (5, 12, 19, 26, or 33). The formation and stability of the coiled-coils were determined by circular dichroism studies in the absence and presence of guanidine hydrochloride. All the proteins with an alanine substituted at position a have a similar stability ([Gdn.HCl]1/2 ranges from 2.6 to 2.9 M), while all the proteins with an alanine substituted at position d have similar stability ([Gdn.HCl]1/2 ranges from 3.6 to 4.2 M), except for the proteins with an alanine substituted in the C-terminal heptad. The greater decrease in stability observed for a Leu----Ala mutation at position a (the average delta delta Gu value is 3.3 kcal/mol) compared to those where the substitution was effected at position d (the average delta delta Gu value is 2.0 kcal/mol) indicates that an Ala mutation at position a has a greater effect on the side-chain packing and hydrophobic interactions in the coiled-coil than an Ala mutation at position d.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intraspecies diversity of the industrial yeast strains Saccharomyces cerevisiae and Saccharomyces pastorianus based on analysis of the sequences of the internal transcribed spacer (ITS) regions and the D1/D2 region of 26S rDNA

We divided industrial yeast strains of Saccharomyces cerevisiae into three groups based on the sequences of their internal transcribed spacer (ITS) regions. One group contained sake yeasts, shochu yeasts, and one bakery yeast, another group contained wine yeasts, and the third group contained beer and whisky yeasts, including seven bakery yeasts. The three groups were distinguished by polymorphisms at two positions, designated positions B and C, corresponding to nucleotide numbers 279 and 301 respectively in the S288C strain. The yeasts in the Japanese group had one thymine at position B and one thymine at position C. The wine yeasts had one thymine at position B and one cytosine at position C. And the beer and whisky yeasts had two thymines at position B and one cytosine at position C. Strains of S. pastorianus were divided into three groups based on the sequences of their 26S rDNA D1/D2 and ITS regions.     

Changing the location of the Schiff base counterion in rhodopsin.

Rhodopsin and all of the vertebrate visual pigments have a carboxylic acid residue, Glu113, in the third transmembrane segment that serves as a counterion to the protonated Schiff base nitrogen of the chromophore. We show here that the counterion in bovine rhodopsin can be moved from position 113 to 117 without significantly changing the wild-type spectral properties of the protein. A series of double mutants were constructed where the Glu113 counterion was changed to Gln and an Asp residue was substituted for amino acid residues from position 111 to 121 in the third transmembrane segment of the protein. Only at position 117 can an Asp fully substitute for the counterion at position 113. The double mutant E113Q,-A117D has an absorption maximum at 493 nm which is independent of pH in the range 5.6-8.4 and independent of the presence of external chloride anions. An Asp at no other position tested in the third transmembrane segment can fully substitute for the Glu counterion at position 113. Partial substitution is observed for an Asp at position 120. Residues 113, 117, and 120 are expected to lie along the same face of an alpha-helix. These results suggest that the Schiff base nitrogen in rhodopsin is located between residues 113 and 117 but there is enough flexibility in the protein to allow partial interaction with an Asp at position 120. Position 117 is the same location of the counterion in the related biogenic amine receptors.     

Characterization of the active-site residues asparagine 167 and lysine 161 of the IMP-1 metallo beta-lactamase

The roles of lysine at position 161 and asparagine at position 167 in IMP-1 metallo beta-lactamase were studied by site-directed mutagenesis. These residues are highly conserved in metallo beta-lactamases and are thought to be present in the active-site cavity. Mutant enzymes with alanine or aspartic acid at position 167 showed almost the same properties as the wild-type enzyme. Kinetic parameters for the mutant enzymes differing at position 161 indicated that the positive charge of lysine 161 is required for electrostatic interaction with the carboxyl moiety of the substrate, i.e. C-3 of penicillins or C-4 of cephalosporins.     

Enzymatic conversion of adenosine to inosine in the wobble position of yeast tRNAAsp: the dependence on the anticodon sequence.

We have investigated the specificity of the tRNA modifying enzyme that transforms the adenosine at position 34 (wobble position) into inosine in the anticodon of several tRNAs. For this purpose, we have constructed sixteen recombinants of yeast tRNAAsp harboring an AXY anticodon (where X or Y was one of the four nucleotides A, G, C or U). This was done by enzymatic manipulations in vitro of the yeast tRNAAsp, involving specific hydrolysis with S1-nuclease and RNAase A, phosphorylation with T4-polynucleotide kinase and ligation with T4-RNA ligase: it allowed us to replace the normal anticodon GUC by trinucleotides AXY and to introduce simultaneously a 32P-labelled phosphate group between the uridine at position 33 and the newly inserted adenosine at position 34. Each of these 32P-labelled AXY "anticodon-substituted" yeast tRNAAsp were microinjected into the cytoplasm of Xenopus laevis oocytes and assayed for their capacity to act as substrates for the A34 to I34 transforming enzyme. Our results indicate that: 1/ A34 in yeast tRNAAsp harboring the arginine anticodon ACG or an AXY anticodon with a purine at position 35 but with A, G or C but not U at position 36 were efficiently modified into I34; 2/ all yeast tRNAAsp harboring an AXY anticodon with a pyrimidine at position 35 (except ACG) or uridine at position 36 were not modified at all. This demonstrates a strong dependence on the anticodon sequence for the A34 to I34 transformation in yeast tRNAAsp by the putative cytoplasmic adenosine deaminase of Xenopus laevis oocytes.     

The residue composition of the aromatic anchor of the second transmembrane helix determines the signaling properties of the aspartate/maltose chemoreceptor Tar of Escherichia coli

Repositioning of the tandem aromatic residues (Trp-209 and Tyr-210) at the cytoplasmic end of the second transmembrane helix (TM2) modulates the signal output of the aspartate/maltose chemoreceptor of Escherichia coli (Tar(Ec)). Here, we directly assessed the effect of the residue composition of the aromatic anchor by studying the function of a library of Tar(Ec) variants that possess all possible combinations of Ala, Phe, Tyr, and Trp at positions 209 and 210. We identified three important properties of the aromatic anchor. First, a Trp residue at position 209 was required to maintain clockwise (CW) signal output in the absence of adaptive methylation, but adaptive methylation restored the ability of all of the mutant receptors to generate CW rotation. Second, when the aromatic anchor was replaced with tandem Ala residues, signaling was less compromised than when an Ala residue occupied position 209 and an aromatic residue occupied position 210. Finally, when Trp was present at position 209, the identity of the residue at position 210 had little effect on baseline signal output or aspartate chemotaxis, although maltose taxis was significantly affected by some substitutions at position 210. All of the mutant receptors we constructed supported some level of aspartate and maltose taxis in semisolid agar swim plates, but those without Trp at position 209 were overmethylated in their baseline signaling state. These results show the importance of the cytoplasmic aromatic anchor of TM2 in maintaining the baseline Tar(Ec) signal output and responsiveness to attractant signaling.     

Fluorescence spectroscopy as a probe of the effect of phosphorylation at serine 40 of tyrosine hydroxylase on the conformation of its regulatory domain

Phosphorylation of Ser40 in the regulatory domain of tyrosine hydroxylase activates the enzyme by increasing the rate constant for dissociation of inhibitory catecholamines from the active site by 3 orders of magnitude. To probe the changes in the structure of the N-terminal domain upon phosphorylation, individual phenylalanine residues at positions 14, 34, and 74 were replaced with tryptophan in a form of the protein in which the endogenous tryptophans had all been mutated to phenylalanine (W(3)F TyrH). The steady-state fluorescence anisotropy of F74W W(3)F TyrH was unaffected by phosphorylation, but the anisotropies of both F14W and F34W W(3)F TyrH increased significantly upon phosphorylation. The fluorescence of the single tryptophan residue at position 74 was less readily quenched by acrylamide than those at the other two positions; fluorescence increased the rate constant for quenching of the residues at positions 14 and 34 but did not affect that for the residue at position 74. Frequency domain analyses were consistent with phosphorylation having no effect on the amplitude of the rotational motion of the indole ring at position 74, resulting in a small increase in the rotational motion of the residue at position 14 and resulting in a larger increase in the rotational motion of the residue at position 34. These results are consistent with the local environment at position 74 being unaffected by phosphorylation, that at position 34 becoming much more flexible upon phosphorylation, and that at position 14 becoming slightly more flexible upon phosphorylation. The results support a model in which phosphorylation at Ser40 at the N-terminus of the regulatory domain causes a conformational change to a more open conformation in which the N-terminus of the protein no longer inhibits dissociation of a bound catecholamine from the active site.     

Analysis of the plasticity of location of the Arg244 positive charge within the active site of the TEM‐1 β‐lactamase

A large number of β‐lactamases have emerged that are capable of conferring bacterial resistance to β‐lactam antibiotics. Comparison of the structural and functional features of this family has refined understanding of the catalytic properties of these enzymes. An arginine residue present at position 244 in TEM‐1 β‐lactamase interacts with the carboxyl group common to penicillin and cephalosporin antibiotics and thereby stabilizes both the substrate and transition state complexes. A comparison of class A β‐lactamase sequences reveals that arginine at position 244 is not conserved, although a positive charge at this structural location is conserved and is provided by an arginine at positions 220 or 276 for those enzymes lacking arginine at position 244. The plasticity of the location of positive charge in the β‐lactamase active site was experimentally investigated by relocating the arginine at position 244 in TEM‐1 β‐lactamase to positions 220, 272, and 276 by site‐directed mutagenesis. Kinetic analysis of the engineered β‐lactamases revealed that removal of arginine 244 by alanine mutation reduced catalytic efficiency against all substrates tested and restoration of an arginine at positions 272 or 276 partially suppresses the catalytic defect of the Arg244Ala substitution. These results suggest an evolutionary mechanism for the observed divergence of the position of positive charge in the active site of class A β‐lactamases.     

Potassium Channel Gating in the Absence of the Highly Conserved Glycine of the Inner Transmembrane Helix

Potassium channel activation regulates cellular excitability, such as in neuronal and cardiac cells. Regulation of ion channel activity relies on a switching mechanism between two major conformations, the open and closed states, known as gating. It has been suggested that potassium channels are generally gated via a pivoted mechanism of the pore-lining helix (TM2) in the proximity of a glycine that is conserved in about 80% of potassium channels, even though about 20% of the channels lack a glycine at this position. Yet, as we show in G-protein gated potassium (Kir3) channels that lack a glycine at this position, the βγ subunits of G-proteins can still stimulate channel activity. Our results suggest that the effect of mutation of the central glycine (at position 175 in Kir3.4) on βγ-induced whole-cell currents is related to the extent of the interaction between residues located at the position of the central glycine and two residues, one located in the signature sequence of the selectivity filter (T149 in Kir3.4) and the other in the pore helix (E147 in Kir3.4). Our results also suggest that interactions with position 149 are more detrimental to channel function than interactions with position 147. The ability of Gβγ to overcome such restraining interactions is likely to depend on a combination of characteristics specific to each residue.     

Correlation of structure with transforming activity of the P21 proteins with substitutions at positions 12 and 16

The structural effects of amino acid substitutions at positions 12 and 16 in the amino-terminal segment (Tyr 4-Ala 18) of the ras-oncogene-encoded P21 proteins have been investigated using conformational energy analysis. The P21 protein with Val at position 12 and Lys at position 16 is known to have high transforming ability, while the P21 protein with Val at position 12 and Asn at position 16 is known to have poor transforming ability, similar to that of the normal protein (with Gly at 12 and Lys at 16.) The current results demonstrate a significant conformational change at position 15 induced by the substitution of Asn for Lys at position 16, which could explain this alteration in transformation potential. These findings are consistent with previous results suggesting the existence of a normal and a malignancy-causing conformation for the P21 proteins and suggest that the critical transforming region may encompass residues 12–15.     

Insertion of an electronegative sulfur atom in the side chain of position 5 of angiotensin II: changes in the tachyphylactic properties of the peptide.

Angiotensin II (AII) analogs bearing n-Leu, Met or S-substituted groups for cysteine at position 5 were studied regarding their agonistic and tachyphylactic properties. It was shown that these analogs lowered the relative affinity towards the AT1 receptor as determined by contractile responses, which could be due to the removal of the beta-branching residue at position 5. Insertion of a sulfur atom in a different position away from the attached backbone carbon atom presented no significant difference in EC50 values for these analogs. Interestingly, the S-bearing analogs at position 5 were full agonists but the tachyphylactic property was lost, in contrast to [n-Leu5]AII, which still induced reduction of the contractile responses. Nevertheless after replacing the Asp with Sar in position 1 (Sar1) tachyphylaxis was again established. It is concluded that the insertion of Met or an S-substituted cysteine into the side chain at position 5 of AII may promote interactions with its receptor due to the slight electronegative character of the sulfur atom and changes in the restricted conformational freedom of the Ile5 residue in the AII molecule. This was overcome by Sar1, probably through interactions due to its fully protonated N-terminal amino group and favoring the conformation responsible for the tachyphylaxis phenomenon.     

Site-directed mutagenesis of Pro327 in the lac permease of Escherichia coli

By use of oligonucleotide-directed, site-specific mutagenesis, Pro327 in the lac permease of Escherichia coli has been replaced with Ala, Gly, or Leu. Permease with Ala at position 327 catalyzes lactose/H+ symport in a manner indistinguishable from wild-type permease. Permease with Gly at position 327, on the other hand, exhibits about one-tenth the activity of wild-type permease but catalyzes lactose accumulation to essentially the same steady-state level as wild-type permease. Finally, permease with Leu at position 327 is completely inactive. The results demonstrate that there is no relationship between permease activity and the helix-breaking (Pro and Gly) or helix-making (Ala and Leu) properties of the residue at position 327. It is suggested that it is primarily a chemical property of the side chain at position 327 (i.e., bulk, hydropathy, and/or ability to hydrogen bond) that is critical for activity and that neither cis/trans isomerization of Pro327 nor the presence of a kink at this position is important.     

Molecular analysis of kanamycin and viomycin resistance in Mycobacterium smegmatis by use of the conjugation system.

We examined the molecular mechanisms of resistance to kanamycin and viomycin in Mycobacterium smegmatis. All of the M. smegmatis strains with high-level kanamycin resistance had a nucleotide substitution from A to G at position 1389 of the 16S rRNA gene (rrs). This position is equivalent to position 1408 of Escherichia coli, and mutation at this position is known to cause aminoglycoside resistance. Mutations from G to A or G to T at position 1473 of the M. smegmatis rrs gene were found in viomycin-resistant mutants which had been designated vicB mutants in our earlier studies. Using the M. smegmatis conjugation system, we confirmed that these mutations indeed contributed to kanamycin and viomycin resistance, and kanamycin susceptibility was dominant over resistance in a heterogenomic strain. Additional experiments showed that three of four Mycobacterium tuberculosis strains with high-level kanamycin resistance had a mutation from A to G at position 1400, which was equivalent to position 1389 of M. smegmatis.     

Framework residue 71 is a major determinant of the position and conformation of the second hypervariable region in the VH domains of immunoglobulins

Analysis of the immunoglobulins of known structure reveals systematic differences in the position and main-chain conformation of the second hypervariable region of the VH domain (H2). We show that the major determinant of the position of H2 is the size of the residue at site 71, a site that is in the conserved framework of the VH domain. It is likely that for about two thirds of the known VH sequences the size of the residue at this site is also a major determinant of the conformation of H2. This effect can override the predisposition of the sequence, as in the case of the H2 loop of J539, which is an exception to the rules relating sequence and conformation of short hairpin loops. Understanding the relationship between the residue at position 71 and the position and conformation of H2 has applications to the prediction and engineering of antigen-binding sites of immunoglobulins.     

Effects of mutations at glycine residues in the hydrophobic region of the Escherichia coli prolipoprotein signal peptide on the secretion across the membrane

Each of the 2 glycine residues in the hydrophobic region of the prolipoprotein signal peptide of Escherichia coli was systematically deleted or substituted with a valine residue by oligonucleotide-directed site-specific mutagenesis. Functional analysis of four such mutants as well as four double mutants, resulting from combinations of any two of the single mutations, revealed that (a) glycine residues at positions 9 and 14 could be replaced individually or at the same time with a valine residue without affecting the secretion of prolipoprotein; (b) the deletion of glycine at position 9 had no effect on the secretion of prolipoprotein whereas, when glycine at position 14 was deleted, the glyceride modification and the processing of the mutant prolipoprotein occurred at a much slower rate at 42 degrees C than those of the wild type prolipoprotein; and (c) the effects of deleting glycine at position 14 could be suppressed by the deletion of glycine at position 9, which resulted in shortening the hydrophobic region of the prolipoprotein signal peptide by 2 amino acid residues. These results indicate that the hydrophobic region of the prolipoprotein signal peptide has remarkable flexibility in terms of the relationship between its primary structure and function in protein secretion.     

Functional analysis of the residues C770 and G771 of E. coli 16S rRNA implicated in forming the intersubunit bridge B2c of the ribosome

Structural analyses have shown that nucleotides at the positions 770 and 771 of Escherichia coli 16S rRNA are implicated in forming one of highly conserved intersubunit bridges of the ribosome, B2c. To examine a functional role of these residues, base substitutions were introduced at these positions and mutant ribosomes were analyzed for their protein synthesis ability using a specialized ribosome system. The results showed requirement of a pyrimidine at the position 770 for ribosome function regardless of the nucleotide identity at the position 771. Sucrose gradient profiles of ribosomes revealed that the loss of protein-synthesis ability of mutant ribosome bearing a base substitution from C to G at the position 770 stems from its inability to form 70S ribosomes. These findings indicate involvement of nucleotide at the position 770, not 771, in ribosomal subunit association and provide a useful rRNA mutation that can be used as a target to investigate the physical interaction between 16S and 23S rRNA.     

Mechanism underlying the inner membrane retention of Escherichia coli lipoproteins caused by Lol avoidance signals

Escherichia coli lipoproteins are localized to either the inner or outer membrane depending on the residue at position 2. The inner membrane retention signal, Asp at position 2 in combination with certain residues at position 3, functions as a Lol avoidance signal, i.e. the signal inhibits the recognition of lipoproteins by LolCDE that releases lipoproteins from the inner membrane. To understand the role of the residue at position 2, outer membrane-specific lipoproteins with Cys at position 2 were subjected to chemical modification followed by the release reaction in reconstituted proteoliposomes. Sulfhydryl-specific introduction of nonprotein molecules or a negative charge to Cys did not inhibit the LolCDE-dependent release. In contrast, oxidation of Cys to cysteic acid resulted in generation of the Lol avoidance signal, indicating that the Lol avoidance signal requires a critical length of negative charge at the second residue. Furthermore, not only modification of the carboxylic acid of Asp at position 2 but also that of the amine of phosphatidylethanolamine abolished the Lol avoidance function. Based on these results, the Lol avoidance mechanism is discussed.     

Neuronal processing delays are compensated in the sensorimotor branch of the visual system

Moving objects change their position until signals from the photoreceptors arrive in the visual cortex. Nonetheless, motor responses to moving objects are accurate and do not lag behind the real-world position. The questions are how and where neural delays are compensated for. It was suggested that compensation is achieved within the visual system by extrapolating the position of moving objects. A visual illusion supports this idea: when a briefly flashed object is presented in the same position as a moving object, it appears to lag behind. However, moving objects do not appear ahead of their final or reversal points. We investigated a situation where participants localized the final position of a moving stimulus. Visual perception and short-term memory of the final target position were accurate, but reaching movements were directed toward future positions of the target beyond the vanishing point. Our results show that neuronal latencies are not compensated for at early stages of visual processing, but at a late stage when retinotopic information is transformed into egocentric space used for motor responses. The sensorimotor system extrapolates the position of moving targets to allow for precise localization of moving targets despite neuronal latencies.