PubMed Health⌕ Search

Biomedical subjects

P Gollnick

Publications and source records attributed to P Gollnick.

At least 19 recordsLinked to original sources
PubMed2001

Using nucleotide analogs to probe protein-RNA interactions.

Synthetic nucleotide analogs provide the opportunity to evaluate the importance of individual functional groups on the RNA in protein-RNA complexes. The general approach is to incorporate analogs at a defined position(s) in the RNA target and to evaluate the effect of this substitution on the thermodynamic stability of the protein-RNA complex. The underlying assumption is that if the presence of the analog reduces the stability of the complex, then the functional groups that are altered in the analog interact with the protein. Here we describe the protocols for incorporation of nucleotide analogs either by in vitro transcription using T7 RNA polymerase or by synthetic chemistry. We also describe how we have used this approach to study the interaction of the TRAP protein from Bacillus subtilis with its cognate RNAs consisting of 11 repeats of GAG and/or UAG triplets. By comparing the results of these analog studies with the crystal structure of TRAP bound to an RNA containing 11 GAG repeats, we are able to see that all the functional groups identified by analogs forge direct interactions with the protein. Analog studies also correctly identified residues that do not contact the protein. Moreover, analogs can have indirect effects on the complex stability by altering the structural properties of the RNA.

Bacillus subtilis↗
PubMed2001

The mechanism of RNA binding to TRAP: initiation and cooperative interactions.

The trp RNA-binding Attenuation Protein (TRAP) from Bacillus subtilis is an 11-subunit protein that binds a series of 11 GAG and UAG repeats separated by two to three-spacer nucleosides in trp leader mRNA. The structure of TRAP bound to an RNA containing 11 GAG repeats shows that the RNA wraps around the outside of the protein ring with each GAG interacting with the protein in nearly identical fashion. The only direct hydrogen bond interactions between the protein and the RNA backbone are to the 2'-hydroxyl groups on the third G of each repeat. Replacing all 11 of these guanosines with deoxyriboguanosine eliminates measurable binding to TRAP. In contrast, a single riboguanosine in an otherwise entirely DNA oligonucleotide dramatically stabilizes TRAP binding, and facilitates the interaction of the remaining all-DNA portion with the protein. Studies of TRAP binding to RNAs with between 2 and 11 GAGs, UAGs, AAGs, or CAGs showed that the stability of a TRAP-RNA complex is not directly proportional to the number of repeats in the RNA. These studies also showed that the effect of the identity of the residue in the first position of the triplet, with regard to binding to TRAP, is dependent on the number of repeats in the RNA. Together these data support a model in which TRAP binds to RNA by first forming an initial complex with a small subset of the repeats followed by a cooperative interaction with the remaining triplets.

5' Untranslated Regions↗
PubMed1999

Structure of the trp RNA-binding attenuation protein, TRAP, bound to RNA.

The trp RNA-binding attenuation protein (TRAP) regulates expression of the tryptophan biosynthetic genes of several bacilli by binding single-stranded RNA. The binding sequence is composed of eleven triplet repeats, predominantly GAG, separated by two or three non-conserved nucleotides. Here we present the crystal structure of a complex of TRAP and a 53-base single-stranded RNA containing eleven GAG triplets, revealing that each triplet is accommodated in a binding pocket formed by beta-strands. In the complex, the RNA has an extended structure without any base-pairing and binds to the protein mostly by specific protein-base interactions. Eleven binding pockets on the circular TRAP 11-mer form a belt with a diameter of about 80 A. This simple but elegant mechanism of arresting the RNA segment by encircling it around a protein disk is applicable to both transcription, when TRAP binds the nascent RNA, and to translation, when TRAP binds the same sequence within a non-coding leader region of the messenger RNA.

Bacterial Proteins↗
PubMed1999

Regulatory features of the trp operon and the crystal structure of the trp RNA-binding attenuation protein from Bacillus stearothermophilus.

Characterization of both the cis and trans -acting regulatory elements indicates that the Bacillus stearothermophilustrp operon is regulated by an attenuation mechanism similar to that which controls the trp operon in Bacillus subtilis. Secondary structure predictions indicate that the leader region of the trp mRNA is capable of folding into terminator and anti- terminator RNA structures. B. stearothermophilus also encodes an RNA-binding protein with 77% sequence identity with the RNA-binding protein (TRAP) that regulates attenuation in B. subtilis. The X-ray structure of this protein has been determined in complex with L-tryptophan at 2.5 A resolution. Like the B. subtilis protein, B. stearothermophilus TRAP has 11 subunits arranged in a ring-like structure. The central cavities in these two structures have different sizes and opposite charge distributions, and packing within the B. stearothermophilus TRAP crystal form does not generate the head-to-head dimers seen in the B. subtilis protein, suggesting that neither of these properties is functionally important. However, the mode of L-tryptophan binding and the proposed RNA binding surfaces are similar, indicating that both proteins are activated by l -tryptophan and bind RNA in essentially the same way. As expected, the TRAP:RNA complex from B. stearothermophilus is significantly more thermostable than that from B. subtilis, with optimal binding occurring at 70 degrees C.

Amino Acid Sequence↗
PubMed1999

Probing the TRAP-RNA interaction with nucleoside analogs.

The trp RNA-binding Attenuation Protein (TRAP) from Bacillus subtilis binds a series of GAG and UAG repeats separated by 2-3 nonconserved spacer nucleotides in trp leader mRNA. To identify chemical groups on the RNA required for stability of the TRAP-RNA complex, we introduced several different nucleoside analogs into each pentamer of the RNA sequence 5'-(UAGCC)-3' repeated 11 times and measured their effect on the TRAP-RNA interaction. Deoxyribonucleoside substitutions revealed that a 2'-hydroxyl group (2'-OH) is required only on the guanosine occupying the third residue of the RNA triplets for high-affinity binding to TRAP. The remaining hydroxyl groups are dispensable. Base analog substitutions identified all of the exocyclic functional groups and N1 nitrogens of adenine and guanine in the second and third nucleotides, respectively, of the triplets as being involved in binding TRAP. In contrast, none of the substitutions made in the first residue caused any detectable changes in affinity, indicating that elements of these bases are not necessary for complex formation and stability. Studies using abasic nucleotides in the first residue of the triplets and in the two spacer residues confirmed that the majority of the specificity and stability of the TRAP-RNA complex is provided by the AG dinucleotide of the triplet repeats. In addition to direct effects on binding, we demonstrate that the N7-nitrogen of adenosine and guanosine in UAG triplet and the 2'-OHs of (UAGCC)11 RNA are involved in the formation of an as yet undetermined structure that interferes with TRAP binding.

Bacillus subtilis↗
PubMed1998

RNA structure inhibits the TRAP (trp RNA-binding attenuation protein)-RNA interaction.

TRAP (trp RNA-binding attenuation protein) regulates expression of the tryptophan biosynthetic genes in response to tryptophan in Bacillus subtilis by binding to two sites containing a series of 9 or 11 (G/U)AG triplet repeats that are generally separated by two or three spacer nucleotides. Previous mutagenesis experiments have identified three TRAP residues, Lys-37, Lys-56, and Arg-58 that are essential for RNA binding. The location of these residues on the TRAP oligomer supports the proposal that RNA binds TRAP by encircling the TRAP oligomer. In this work, we show that RNAs containing 11 GAG or UAG repeats separated by CC dinucleotide spacers (((G/U)AGCC)11) form stable structures that inhibit binding to TRAP. This conclusion is based on the effects of temperature and Mg2+ on the affinity of TRAP for RNAs with CC spacers combined with UV hyperchromicity and circular dichroism. Furthermore, introducing the base analogue 7-deazaguanosine in the ((G/U)AGCC)11 RNAs stabilized the TRAP-RNA interaction. This effect was associated with decreased stability of the RNA structure as measured by circular dichroism spectroscopy. The precise nature of the structure of the ((G/U)AGCC)11 RNAs is not known but evidence is presented that it involves noncanonical interactions. We also observed that substitution of Arg-58 with Lys further reduced the ability of TRAP to interact with structured RNAs. Since in vivo function of TRAP may involve binding to structured RNAs, we suggest a potential function for this residue, which is conserved in TRAP from three different bacilli.

Bacillus↗
PubMed1997

The trp RNA-binding attenuation protein (TRAP) from Bacillus subtilis binds to unstacked trp leader RNA.

TRAP (trp RNA-binding attenuation protein) is a tryptophan-activated RNA-binding protein that regulates expression of the trp biosynthetic genes by binding to a series of GAG and UAG trinucleotide repeats generally separated by two or three spacer nucleotides. Previously, we showed that TRAP contains 11 identical subunits arranged in a symmetrical ring. Based on this structure, we proposed a model for the TRAP.RNA interaction where the RNA wraps around the protein with each repeat of the RNA contacting one or a combination of two adjacent subunits of the TRAP oligomer. Here, we have shown that RNAs selected in vitro based on their ability to bind tryptophan-activated TRAP contain multiple G/UAG repeats and show a strong bias for pyrimidines as the spacer nucleotides between these repeats. The affinity of the TRAP.RNA interaction displays a nonlinear temperature dependence, increasing between 5 degrees C and 47 degrees C and then decreasing from 47 degrees C to 67 degrees C. Differential scanning calorimetry and circular dichroism spectroscopy demonstrate that TRAP is highly thermostable with few detectable changes in the structure between 25 degrees C and 70 degrees C, suggesting that the temperature dependence of this interaction reflects changes in the RNA. Results from circular dichroism and UV absorbance spectroscopy support this hypothesis, demonstrating that trp leader RNA becomes unstacked upon binding TRAP. We propose that the bias toward pyrimidines in the spacer nucleotides of the in vitro selected RNAs represents the inability of Us and Cs to form stable base stacking interactions, which allows the flexibility needed for the RNA to wrap around the TRAP oligomer.

Bacillus subtilis↗
PubMed1997

Alanine-scanning mutagenesis of Bacillus subtilis trp RNA-binding attenuation protein (TRAP) reveals residues involved in tryptophan binding and RNA binding.

In Bacillus subtilis, expression of the trp genes is negatively regulated by an RNA binding protein called TRAP (trp RNA-binding Attenuation Protein), which is activated to bind RNA by binding l-tryptophan. TRAP contains 11 identical subunits assembled in a symmetric ring. We have used alanine-scanning mutagenesis to analyze the functions of surface amino acid residues of TRAP. The in vivo regulatory activity of each mutant TRAP was analyzed in a B. subtilis reporter strain containing a trpE'-'lacZ fusion. Mutant TRAP proteins with defective in vivo regulatory activities were characterized in vitro by measuring their tryptophan binding and RNA binding activities. Most of the mutant proteins with altered tryptophan binding, either affinity or cooperativity, contained substituted residues located on two loops formed by residues 25 to 33 and residues 49 to 52, as well as on the beta-strand and beta-turn contiguous with these loops. Substitution of three residues (Lys37, Lys56 and Arg58) with alanine resulted in significant decreases in the RNA binding activity of TRAP without altering tryptophan binding. Structural analysis shows that these three residues are directly aligned on the outer edge of TRAP. Further mutagenic analysis of these three residues revealed that only lysine or arginine residues at positions 37 or 58 allow proper TRAP function, whereas at position 56, only lysine is functional. Residue Asn20 is the only other residue in TRAP that is located on the line formed by residues 37, 56 and 58, and virtually any amino acid residue is functional at position 20. We propose that RNA wraps around TRAP by interacting with residues Lys37, Lys56 and Arg58.

Alanine↗
PubMed1997

The crystal structure of Citrobacter freundii tyrosine phenol-lyase complexed with 3-(4'-hydroxyphenyl)propionic acid, together with site-directed mutagenesis and kinetic analysis, demonstrates that arginine 381 is required for substrate specificity.

The X-ray structure of tyrosine phenol-lyase (TPL) complexed with a substrate analog, 3-(4'-hydroxyphenyl)propionic acid, shows that Arg 381 is located in the substrate binding site, with the side-chain NH1 4.1 A from the 4'-OH of the analog. The structure has been deduced at 2.5 A resolution using crystals that belong to the P2(1)2(1)2 space group with a = 135.07 A, b = 143.91 A, and c = 59.80 A. To evaluate the role of Arg 381 in TPL catalysis, we prepared mutant proteins replacing arginine with alanine (R381A), with isoleucine (R381I), and with valine (R381V). The beta-elimination activity of R381A TPL has been reduced by 10(-4)-fold compared to wild type, whereas R381I and R381V TPL exhibit no detectable beta-elimination activity with L-tyrosine as substrate. However, R381A, R381I, and R381V TPL react with S-(o-nitrophenyl)-L-cysteine, beta-chloro-L-alanine, O-benzoyl-L-serine, and S-methyl-L-cysteine and exhibit k(cat) and k(cat)/Km values comparable to those of wild-type TPL. Furthermore, the Ki values for competitive inhibition by L-tryptophan and L-phenylalanine are similar for wild-type, R381A, and R381I TPL. Rapid-scanning-stopped flow spectroscopic analyses also show that wild-type and mutant proteins can bind L-tyrosine and form quinonoid complexes with similar rate constants. The binding of 3-(4'-hydroxyphenyl)propionic acid to wild-type TPL decreases at high pH values with a pKa of 8.4 and is thus dependent on an acidic group, possibly Arg404, which forms an ion pair with the analog carboxylate, or the pyridoxal 5'-phosphate Schiff base. R381A TPL shows only a small decrease in k(cat)/Km for tyrosine at lower pH, in contrast to wild-type TPL, which shows two basic pKas with an average value of about 7.8. Thus, it is possible that Arg 381 is one of the catalytic bases previously observed in the pH dependence of k(cat)/Km of TPL with L-tyrosine [Kiick, D. M., & Phillips. R. S. (1988) Biochemistry 27, 7333-7338], and hence Arg 381 is at least partially responsible for the substrate specificity of TPL.

Arginine↗
PubMed1996

Kinetic and thermodynamic analysis of the interaction between TRAP (trp RNA-binding attenuation protein) of Bacillus subtilis and trp leader RNA.

In Bacillus subtilis, expression of the tryptophan biosynthetic genes is regulated in response to tryptophan by an RNA-binding protein called TRAP (trp RNA-binding attenuation protein). TRAP has been shown to contain 11 identical subunits arranged in a symmetrical ring. Kinetic and thermodynamic parameters of the interaction between tryptophan-activated TRAP and trp leader RNA were studied. Results from glycerol gradients and mobility shift gels indicate that two TRAP 11-mers bind to each trp leader RNA. A filter binding assay was used to determine an apparent binding constant of 8.0 +/- 1.3 x 10(9) m-1 (Kd = 0.12 +/- 0.02 nM) for TRAP and an RNA containing residues +36 to +92 of the trp leader RNA in 1 mM L-tryptophan at 37 degrees C. The temperature dependence of Kapp was somewhat unexpected demonstrating that the delta H of the interaction is highly unfavorable at + 15.9 kcal mol-1. Therefore, the interaction is completely driven by a delta S of +97 cal mol-1 K-1. The interaction between tryptophan-activated TRAP and trp leader RNA displayed broad salt and pH activity profiles. Finally, the rate of RNA dissociation from the RNA-TRAP.tryptophan ternary complex was found to be very slow in high concentrations of tryptophan (> 40 microM) but increased in lower tryptophan concentrations. This suggests that dissociation of tryptophan from the ternary complex is the rate-limiting step in RNA dissociation.

Bacillus subtilis↗
PubMed1995

The structure of trp RNA-binding attenuation protein.

The crystal structure of the trp RNA-binding attenuation protein of Bacclius subtilis solved at 1.8 A resolution reveals a novel structural arrangement in which the eleven subunits are stabilized through eleven intersubunit beta-sheets to form a beta-wheel with a large central hole. The nature of the binding of L-tryptophan in clefts between adjacent beta-sheets in the beta-wheel suggests that this binding induces conformational changes in the flexible residues 25-33 and 49-52. It is argued that upon binding, the messenger RNA target forms a matching circle in which eleven U/GAG repeats are bound to the surface of the protein ondecamer modified by the binding of L-tryptophan.

Amino Acid Sequence↗
PubMed1995

Site-directed mutagenesis of His343-->Ala in Citrobacter freundii tyrosine phenol-lyase. Effects on the kinetic mechanism and rate-determining step.

His343 in Citrobacter freundii tyrosine phenol-lyase is conserved in all known sequences of both tyrosine phenol-lyase and tryptophan indole-lyase; it is located near the active-site Lys257 in C. freundii tyrosine phenol-lyase [Antson, A. A., Demidkina, T. V., Gollnick, P., Danter, Z., Von Tersch, R.L., Long, J., Berezhnoy, S. N., Phillips, R. S., Harutyunyan, E. H. & Wilson, K. S. (1993) Biochemistry 32, 4195--4206]. In order to evaluate the role of His343 in the reaction mechanism of tyrosine phenol-lyase and tryptophan indole-lyase, we have mutated it to Ala; the former mutant is referred to as [H343A]tyrosine phenol lyase. All substrates for alpha, beta-elimination (except S-ethyl-L-cysteine) exhibited lower kcat (10-30%) and kcat/Km (1-10%) values with [H343A]tyrosine phenol-lyase than with the wild-type enzyme. The mutant also shows slower rates of deuterium isotope exchange for L-phenylalanine and L-methionine than does the wild type. The pH-dependent behavior in the reaction of 3-fluoro-L-tyrosine with wild-type tyrosine phenol-lyase is identical to that of L-tyrosine described previously [Kiick, D. M. & Phillips, R. S. (1988) Biochemistry 27, 7333-7338]. The pH profile of kcat/Km for this reaction exhibits two pKa values with an average of 7.7 +/- 0.2, indicating that the catalytic mechanism requires two essential basic groups. The pH profile of kcat/Km for 3-fluoro-L-tyrosine with [H343A]tyrosine phenol-lyase also exhibits two pKa values with an average of 7.8 +/- 0.3. However, kcat for 3-fluoro-L-tyrosine is pH-dependent for the mutant, exhibiting two pKa values with an average of about 7.8, whereas it is pH-independent for the wild type. Steady-state kinetic isotope effects on the reactions with wild-type and [H343A]tyrosine phenol-lyase were examined at various pH values. For the wild type, the values of the isotope effects on kcat and kcat/Km for 3-fluoro-L-[alpha-2H]-tyrosine are independent of pH and equal to 3.9 +/- 0.2 and 2.2 +/- 0.3, respectively, while the corresponding values for [H343A]tyrosine phenol-lyase are 5.4 +/- 0.2 and 3.8 +/- 0.3, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Alanine↗
PubMed1995

Translation of trpG in Bacillus subtilis is regulated by the trp RNA-binding attenuation protein (TRAP).

trpG of Bacillus subtilis encodes a glutamine amidotransferase subunit that is involved in the synthesis of both folic acid and L-tryptophan. Expression of trpG is negatively regulated by tryptophan even though this gene is located within a folic acid biosynthetic operon. Examination of both transcriptional and translational gene fusions to lacZ involving trpG and direct measurements of trpG mRNA levels and TrpG polypeptide accumulation demonstrated that translation of trpG is regulated by tryptophan whereas transcription is not. these studies also show that this regulation is mediated by the trp RNA-binding attenuation protein. Deletion and point mutations indicated that regulation is dependent on a series of G/UAG trinucleotide repeats surrounding the putative ribosome-binding site for trpG. Our results are consistent with a model in which the tryptophan-activated trp RNA-binding attenuation protein and ribosomes compete for binding to trpG mRNA.

Amino Acid Sequence↗
PubMed1995

The mtrB gene of Bacillus pumilus encodes a protein with sequence and functional homology to the trp RNA-binding attenuation protein (TRAP) of Bacillus subtilis.

The mtrB gene from Bacillus pumilus encodes a 76-amino-acid polypeptide with 77% identity to the trp RNA-binding attenuation protein (TRAP) from Bacillus subtilis. B. pumilus TRAP binds trp leader RNA from either B. subtilis or B. pumilus in a tryptophan-dependent manner. Altering threonine 52 to alanine eliminated RNA-binding activity of B. pumilus TRAP.

Bacillus↗
PubMed1995

Interaction of the 11-subunit trp RNA-binding attenuation protein (TRAP) with its RNA target.

The trp RNA-binding attenuation protein (TRAP) regulates expression of the B. subtilis trp operon by binding to an RNA target composed of 11 G/UAG repeats in the trp leader transcript. TRAP contains 11 identical subunits arranged in a ring. The binding of 11 molecules of L-tryptophan to sites formed between adjacent subunits activates TRAP to bind trp leader RNA. We propose that the trp leader RNA forms a matching circle on the surface of TRAP when it binds. RNA binding is relatively insensitive to changes in ionic strength and pH, and is apparently driven by changes in entropy. Two lysine residues on the surface of TRAP have been shown to be important for RNA binding.

Bacillus subtilis↗
PubMed1994

11-fold symmetry of the trp RNA-binding attenuation protein (TRAP) from Bacillus subtilis determined by X-ray analysis.

The trp RNA-binding attenuation protein (TRAP) of Bacillus subtilis has been crystallized and examined by crystallography using X-ray synchrotron radiation diffraction data. Crystals of TRAP complexed with L-tryptophan belong to space group C2 with a = 156.8 A, b = 114.05 A, c = 105.9 A, beta = 118.2 degrees. Crystals of a potential heavy-atom derivative of TRAP complexed with 5-bromo-L-tryptophan grow in the same space group with similar cell dimensions. X-ray data for the native crystals and for the derivative have been collected to 2.9 A and 2.2 A resolution, respectively. Peaks in the self-rotation function and in the Patterson synthesis could only be explained by two 11-subunit oligomers (each formed by an 11-fold axis of symmetry) in the asymmetric unit lying with the 11-fold rotation axes parallel to each other. The consequence is that the TRAP molecule has 11-fold symmetry and contains 11 subunits.

Bacillus subtilis↗
PubMed1994

Regulation of the Bacillus subtilis trp operon by an RNA-binding protein.

The Bacillus subtilis tryptophan (trpEDCFBA) operon is regulated by transcription attenuation. Transcription is controlled by two alternative RNA secondary structures, which form in the leader transcript. In the presence of L-tryptophan, a transcription terminator forms and the operon is not expressed, whereas in the absence of tryptophan, an antiterminator structure forms allowing transcription of the operon. The mechanism of selection between these alternative structures involves a trans-acting RNA-binding regulatory protein. This protein is the product of the mtrB gene and is called TRAP for trp attenuation protein. TRAP has been shown to bind specifically to trp leader RNA, and to cause transcription of the trp operon to terminate in the leader region. The model for regulation suggests that in the presence of tryptophan, TRAP binds to the leader RNA and induces formation of the transcription terminator structure, whereas in the absence of tryptophan, the protein does not bind and the antiterminator is formed.

Amino Acid Sequence↗