Biomedical subjects

T Pan

Publications and source records attributed to T Pan.

At least 91 records · Page 5Linked to original sources

PubMed1994

Selection of circularly permuted ribozymes from Bacillus subtilis RNAse P by substrate binding.

The effect of a single break in the phosphodiester backbone of Bacillus subtilis RNAse P RNA (P RNA) was examined using circular permutation analysis (CPA). This method reveals that many of the phosphodiester bonds in this catalytic RNA can be broken with little or no effect on substrate binding. Phosphate positions that show strong effects are located mostly in regions conserved among all RNAse P RNAs, or they are in regions known to interact directly with the pre-tRNA substrate. Two circularly permuted isomers of P RNA were constructed and analyzed in detail. The KM for both circularly permuted isomers is nearly identical to that of the wild-type P RNA. Since the KM of the P RNA is essentially the same as the binding constant to the substrate, this finding confirms the CPA results. The implications of backbone breakage are discussed with respect to folding and catalysis of the RNAse P RNA.

Bacillus subtilis↗

PubMed1994

In vitro selection of small RNAs that bind to Escherichia coli phenylalanyl-tRNA synthetase.

Small RNAs were selected from a highly degenerate library on the basis of their ability to bind tightly to Escherichia coli phenylalanyl-tRNA synthetase (FRS). The 63 nucleotide library consisted of the acceptor stem and portions of the D and T stems of E. coli tRNA(Phe) flanking a 32 nucleotide randomized region. Because FRS binding relies on a correctly folded tRNA substrate, the selected variants from this library were expected to resemble tRNA(Phe) structure. After seven cycles of selection, the RNA library bound to FRS with similar affinity to that of the E. coli tRNA(Phe), but did not show detectable aminoacylation. Fourteen FRS-specific isolates were sequenced and found to contain an anticodon stem-loop including the anticodon triplet of tRNA(Phe). The tight-binding RNAs fell into two classes depending on the location of this step-loop within the sequence. The acceptor stem defined by the non-randomized sequence was also found to be essential for binding. Mutation of two residues within a common hexanucleotide sequence present in one of the classes reduced binding to FRS. Taken together, these results suggest that in order to bind RNAs tightly, FRS requires the simultaneous interaction of the anticodon stem-loop and acceptor stem, and additional sequences needed for proper folding. This approach should assist in the detection of motifs that resemble tRNA, but are too dissimilar to be identified by sequence comparison.

Base Sequence↗

PubMed1994

Properties of an in vitro selected Pb2+ cleavage motif.

The addition of Pb2+ to a small RNA molecule consisting of an asymmetric internal loop of six nucleotides results in site-specific cleavage followed by hydrolysis of the 2',3'-cyclic phosphate intermediate [Pan, T., & Uhlenbeck, O.C. (1992) Nature 358, 560-563]. Here we show that the reaction is highly specific for Pb2+ and the cleavage rate increases exponentially with pH from 5.5 to 7.0, both in the presence and in the absence of Mg2+. This suggests that the reaction mechanism involves Pb2+ hydroxide acting as a base. Several sequence variants of the RNA are found to be equally active in both steps of the reaction, suggesting that they fold into a similar structure.

Cations, Divalent↗

PubMed1994

Structure and function of retinal ganglion cells innervating the cat's geniculate wing: an in vitro study.

We have examined in vitro the morphology and visual response properties of retinal ganglion cells innervating a component of the cat's lateral geniculate nucleus known as the geniculate wing (or retinorecipient zone of the pulvinar). Ganglion cells were first labeled in situ by retrograde transport of fluorescent microspheres from the geniculate wing. Labeled cells were injected intracellular with Lucifer yellow and biocytin in the isolated retina and visualized immunohistochemically. With one exception, stained cells appeared to belong to a single morphological class that corresponded closely to the epsilon cell of earlier descriptions (Leventhal et al., 1980; Rodieck and Watanabe, 1986). They had somas comparable in size to those of beta cells and large, sparse dendritic trees that ramified in the inner (ON) sublayer of the inner plexiform layer. Dendritic fields increased in size with eccentricity, but only within the central retina, and were among the largest so far reported for cat ganglion cells, exceeding those of alpha cells at most eccentricities. Dendritic profiles were typically elliptical with long axes pointing toward the area centralis. Axons were about as thick as those of beta cells and thicker than those of other varieties of non-alpha, non-beta ganglion cells. We recorded extracellularly from microsphere-labeled wing-projecting ganglion cells in a superfused, flattened eyecup preparation. All such cells exhibited sustained responses to standing contrast and had very large, concentric receptive fields with ON-centers and OFF-surrounds. Their response to gratings showed that they have relatively poor spatial resolution and a moderate amount of nonlinearity of spatial summation. These cells thus have many physiological response properties in common with ganglion cells previously termed "on-center tonic W-cells," "on-center sluggish sustained cells," and "Q-cells." These findings indicate that ganglion cells innervating the cat's geniculate wing form a structurally and functionally homogeneous class. Their large dendritic and receptive fields and low-pass spatial frequency tuning suggest that fine spatial resolution is not required for the execution of their functional role(s).

Animals↗

PubMed1993

Using circular permutation analysis to redefine the R17 coat protein binding site.

The bacteriophage R17 coat protein binding site consists of an RNA hairpin with a single purine nucleotide bulge in the helical stem. Circular permutation analysis (CPA) was used to examine binding effects caused by a single break in the phosphodiester backbone. This method revealed that breakage of all but one phosphodiester bond within a well-defined binding site substantially reduced the binding affinity. This is probably due to destabilization of the hairpin structure upon breaking the ribose phosphates at these positions. One circularly permuted isomer with the 5' and 3' ends at the bulged nucleotide bound with wild-type affinity. However, extending the 5' end of this CP isomer greatly reduces binding, making it unlikely that this circularly permuted binding site will be active when embedded in a larger RNA. CPA also locates the 5' and 3' boundaries of protein binding sites on the RNA. The 5' boundary of the R17 coat protein site as defined by CPA was two nucleotides shorter (nucleotides -15 to +2) than the previously determined site (-17 to +2). The smaller binding site was verified by terminal truncation experiments. A minimal-binding fragment (-14 to +2) was synthesized and was found to bind tightly to the coat protein. The site size determined by 3-ethyl-1-nitrosourea-modification interference was larger at the 5' end (-16 to +1), probably due, however, to steric effects of ethylation of phosphate oxygens. Thus, the apparent site size of a protein binding site is dependent upon the method used.

Bacteriophages↗

PubMed1993

Circularly permuted DNA, RNA and proteins--a review.

Circular permutation represents a form of macromolecular isomerization when the normal termini are covalently linked and new termini introduced by breaking the backbone elsewhere. Here, we describe implications of circular permutation on the folding and function of biologically relevant macromolecules. A method permitting the analysis of the folding of all circularly permuted isomers of RNA is presented that has been successfully applied for a tRNA and the binding site of the coliphage R17 coat protein.

Animals↗

PubMed1993

Replacement of RNA hairpins by in vitro selected tetranucleotides.

An in vitro selection method based on the autolytic cleavage of yeast tRNA(Phe) by Pb2+ was applied to obtain tRNA derivatives with the anticodon hairpin replaced by four single-stranded nucleotides. Based on the rates of the site-specific cleavage by Pb2+ and the presence of a specific UV-induced crosslink, certain tetranucleotide sequences allow proper folding of the rest of the tRNA molecule, whereas others do not. One such successful tetramer sequence was also used to replace the acceptor stem of yeast tRNA(Phe) and the anticodon hairpin of E.coli tRNA(Phe) without disrupting folding. These experiments suggest that certain tetramers may be able to replace structurally nonessential hairpins in any RNA.

Base Sequence↗

PubMed1993

A study of homologous chromosomes using a morphometric approach.

A previous study of 100 karyotyped metaphase cells has demonstrated the utility of a graphic arts tool in deriving chromosome measurements for relative length determination. In the present study we utilize this same approach to address the question: "What are the average differences in relative lengths between apparently normal homologous chromosomes?" Normal standards derived from this study will be useful for testing specific hypotheses involving heteromorphic differences between homologs. No such data on normal controls could readily be found in either An International System for Human Cytogenetic Nomenclature (1985) or elsewhere.

Chromosomes↗

PubMed1992

A small metalloribozyme with a two-step mechanism.

An RNA molecule consisting of an asymmetric internal loop of six nucleotides can be rapidly and specifically cleaved by Pb2+ in the presence of Mg2+. The 5' cleavage product terminates with a 3' phosphomonoester generated from a 2',3'-cyclic phosphodiester reaction intermediate. This two-step reaction mechanism resembles that of many protein ribonucleases but has not previously been observed for reactions catalysed by RNA.

Base Sequence↗

PubMed1992

In vitro selection of RNAs that undergo autolytic cleavage with Pb2+.

An in vitro selection method has been developed to obtain RNA molecules that specifically undergo autolytic cleavage reactions by Pb2+ ion. The method utilizes a circular RNA intermediate which is regenerated following the cleavage reaction to allow amplification and multiple cycles of selection. Pb2+ is known to catalyze a specific cleavage reaction between U17 and G18 of yeast tRNA(Phe). Starting from pools of RNA molecules which have a random distribution of sequences at nine or ten selected positions in the sequence of yeast tRNA(Phe), we have isolated many RNA molecules that undergo rapid and specific self-cleavage with Pb2+ at a variety of different sites. Terminal truncation experiments suggest that most of these self-cleaving RNA molecules do not fold like tRNA. However, two of the variants are cleaved rapidly with Pb2+ at U17 even though they lack the highly conserved nucleotides G18 and G19. Both specific mutations and terminal truncation experiments suggest that the D and T loops of these two variants interact in a manner similar to that of tRNA(Phe) despite the absence of the G18U55 and G19C56 tertiary interactions. A model for an alternate tertiary interaction involving a U17U55 pair is presented. This model may be relevant to the structure of about 100 mitochondrial tRNAs that also lack G18 and G19. The selection method presented here can be directly applied to isolate catalytic RNAs that undergo cleavage in the presence of other metal ions, modified nucleotides, or sequence-specific nucleases.

Base Composition↗

PubMed1991

Folding of circularly permuted transfer RNAs.

All of the ribose-phosphate linkages in yeast tRNA(Phe) that could be cleaved without affecting the folding of the molecule have been determined in a single experiment. Circular permutation analysis subjects circular tRNA molecules to limited alkaline hydrolysis in order to generate one random break per molecule. Correctly folded tRNAs were identified by lead cleavage at neutral pH, a well-characterized reaction that requires proper folding of tRNA(Phe). Surprisingly, most of the circularly permuted tRNA molecules folded correctly. This result suggests that the tRNA folding motif could occur internally within other RNA sequences, and a computer search of Genbank entries has identified many examples of such motifs.

Base Sequence↗

PubMed1991

Structure of the binuclear metal-binding site in the GAL4 transcription factor.

The GAL4 transcription factor from yeast contains within its N-terminal DNA-binding domain an amino acid sequence containing six cysteine residues, C11-X2-C14-X6-C21-X6-C28-X2-C31-X6-C38. The six Cys residues will form a binuclear metal cluster with either Zn(II) or Cd(II) in which two of the -S- donors are bridging ligands between the two metal ions. Binding of Zn(II) or Cd(II) to the GAL4 DNA-binding domain is essential to induce the conformation of GAL4 required for the protein to recognize the specific DNA sequence, UASG, to which GAL4 binds. Evidence for the presence of the binuclear cluster has come from 113Cd NMR and 2D 1H-113Cd heteronuclear NMR studies of the cloned DNA-binding domain of GAL4 consisting of the N-terminal 62 residues, GAL4(62*) [Pan and Coleman (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 2077]. Cd(II) binding to the GAL4 DNA is highly cooperative, thus the Cd2Cys6 cluster is always formed. On the other hand, Zn(II) forms well-defined Zn1 and Zn2 complexes with the DNA-binding domain of GAL4, both of which bind specifically to the UASG DNA sequence. The structural details of the Cd2-, Zn2-, and Zn1GAL4(62*) proteins have been determined by a variety of heteronuclear and 2D NMR techniques. When Cd(II) is exchanged for Zn(II), the cluster appears to expand to accommodate the larger Cd(II) ion as suggested by changes of 2 to 4 Hz in the 3JHN alpha coupling constants for the amino acid residues which form the polypeptide loops enclosing the cluster, residues 10-40. These changes suggest alterations in the backbone phi torsional angles of from 20 degrees to 30 degrees. A metal-ligand structure derived from the 1H-113Cd heteronuclear NMR as well as the polypeptide backbone connectivity around the cluster as determined from short-range 1H-1H NOE's is presented. The metal ions also determine the major folding of GAL4(62*), since the chemical shift dispersion in the entire NH-alpha CH fingerprint region of the 1H-1H COSY spectrum collapses on removal of the metal ion. Two short segments of the GAL4(62*) polypeptide (residues 14-19 and 30-36 in the cluster forms, 12-19 and 30-36 in the Zn1 species) show significant dNN(i,i + 1) NOE's. These short segments of polypeptide chain are the only ones that could be helical in the GAL4(62*).(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed1991

Sequential assignments of the 1H NMR resonances of Zn(II)2 and 113Cd(II)2 derivatives of the DNA-binding domain of the GAL4 transcription factor reveal a novel structural motif for specific DNA recognition.

The DNA-binding domain of the GAL4 transcription factor, consisting of the 62 N-terminal amino acid residues and denoted GAL4(62*), contains a novel Zn(II)2Cys6 or Cd(II)2Cys6 binuclear cluster [Pan, T., & Coleman, J. E. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 2077]. Specific DNA recognition requires residues located within as well as C terminal to this binuclear cluster. 1H NMR sequential assignments have been carried out on Zn(II)2- and 113Cd(II)2GAL4(62*) by using DQF-COSY, relayed COSY, double-relayed COSY, and NOESY. The ligands of the two tetrahedral metal-binding sites have been identified as Cys11, Cys14, Cys21, and Cys31 to one metal ion and Cys28, Cys38, Cys21, and Cys31 to the other metal ion with Cys21 and Cys31 as ligands shared between the two metal ions. No alpha-helices can be found within the GAL4(62*) structure, which consists of a series of turns to accommodate the metal cluster, followed by irregular loops and turns from residues 42 to 60, the "specificity region", whose sequence contributes importantly to specific DNA recognition. Long-distance NOE's are observed between residues forming the binuclear cluster and several residues within the specificity region, indicating that the latter is folded compactly onto the metal cluster. The requirement of the Zn(II)2Cys6 binuclear cluster and the specificity region for binding to DNA reveals GAL4 as a member of a class of specific DNA-binding proteins using a new structural motif for the recognition of specific DNA sequences. Specific DNA binding by this class of proteins is achieved by use of turns and loops that enclose a Zn(II)2Cys6 binuclear cluster, instead of alpha-helices or beta-strands as observed in specific DNA-binding proteins described previously.

Amino Acid Sequence↗

PubMed1990

The transcription factor LAC9 from Kluyveromyces lactis-like GAL4 from Saccharomyces cerevisiae forms a Zn(II)2Cys6 binuclear cluster.

The DNA binding domain of the transcription factor LAC9 contains 6 cysteine residues with spacing in the primary peptide sequence identical to that found in the DNA binding domain of the GAL4 transcription factor. In GAL4, the CysX2CysX6CysX6CysX2CysX6Cys motif has been shown to form a Zn(II)2Cys6 binuclear cluster (Pan, T. and Coleman, J. E. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 2077-2081), representing a new structure for a Zn(II)-containing transcription factor which differs from the "zinc finger" motif first described for TFIIIA. LAC9 has been shown to bind two Zn(II) ions (Halvorsen, Y. C., Nandabalan, K., and Dickson, R. D. (1990) J. Biol. Chem. 265, 13283-13289). The similarity of the amino acid sequence and the Cys spacing within the DNA binding domain suggest that LAC9 should also be capable of forming the Zn(II)2Cys6 cluster found in GAL4. A fragment of LAC9 consisting of 144 amino acid residues spanning the DNA binding domain has been prepared with 113Cd(II) substituted for the two native Zn(II) ions. 113Cd NMR of this fragment (denoted LAC9(85-228*] has been carried out in an attempt to test the hypothesis that LAC9, like GAL4, forms a binuclear cluster. The chemical shifts of the two bound 113Cd(II) ions, 705 and 692 ppm respectively, are consistent with ligation of each 113Cd(II) ion to 4 sulfur atoms. The best model for such ligation is that two of the cysteine S- form bridges between the two Cd(II) ions. Formation of a Zn(II)-Cd(II) hybrid form of LAC9(85-228*) has also been observed. We conclude that LAC9 contains a Zn(II)2Cys6 binuclear cluster as previously reported for GAL4.

Cations, Divalent↗

PubMed1990

Cadmium-113 NMR studies of the DNA binding domain of the mammalian glucocorticoid receptor.

The DNA binding domain of the mammalian glucocorticoid hormone receptor (GR) contains nine highly conserved cysteine residues, a conservation shared by the superfamily of steroid and thyroid hormone receptors. A fragment [150 amino acids (AA) in length] consisting of GR residues 407-556, containing within it the entire DNA binding domain (residues 440-525), has been overexpressed and purified from Escherichia coli previously. This fragment has been shown to contain 2.3 +/- 0.2 mol of Zn(II) per mole of protein [Freedman, L. P., Luisi, B. F., Korszun, Z. R., Basavappa, R., Sigler, P. B., & Yamamoto, K. R. (1988) Nature 334, 543]. Zn(II) [or Cd(II) substitution] has been shown to be essential for specific DNA binding. 113Cd NMR of a cloned construct containing the minimal DNA binding domain of 86 AA residues [denoted GR(440-525)] with 113Cd(II) substituted for Zn(II) identifies 2 Cd(II) binding sites by the presence of 2 113Cd NMR signals each of which integrates to 1 113Cd nucleus. The chemical shifts of these two sites, 704 and 710 ppm, suggest that each 113Cd(II) is coordinated to four isolated -S- ligands. Shared -S- ligands connecting the two 113Cd(II) ions do not appear to be present, since their T1s differ by 10-fold, 0.2 and 2.0 s, respectively. Addition of a third 113Cd(II) or Zn(II) to 113Cd2GR(440-525) results in occupancy of a third site, which introduces exchange modulation of the two original 113Cd NMR signals causing them to disappear. Addition of EDTA to the protein restores the original two signals.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1990

The DNA binding domain of GAL4 forms a binuclear metal ion complex.

The transcription factor GAL4 from Saccharomyces cerevisiae requires Zn(II) or Cd(II) for specific recognition of the UASG sequence (Pan & Coleman, 1989). An N-terminal fragment consisting of the first 63 amino acid residues of GAL4 [GAL4(63)] has been obtained by partial tryptic proteolysis of a cloned and overproduced N-terminal domain of 149 residues, GAL(149). We show that GAL4(63) contains the minimal GAL4 DNA binding domain. GAL4(63) binds tightly 1-2 mol of Zn(II) or 2 mol of Cd(II). 113Cd NMR of 113Cd(II)-substituted GAL4(63) reveals structural identity between the metal binding domains of GAL4(63) and that of the larger precursor GAL4(149). 113Cd(II) can be substituted for the Zn(II) in GAL4(63), and two 113Cd NMR signals are observed at 706 and 669 ppm, both suggesting coordination of 113Cd(II) to three or four -S- ligands. With the exception of the N-terminal methionine, the only sulfur-containing residues are the six highly conserved cysteines. High-resolution 1H NMR of Zn(II)-GAL4(63) and Cd(II)-GAL4(63) show the two proteins to have almost identical conformations and to be present as monomers in solutions up to millimolar concentration. This leads us to postulate that GAL4 does not possess a TFIIIA-like "Zn-finger" but forms a binuclear metal cluster involving all six cysteines in a "cloverleaf"-like array. GAL4(63) contains about 60% alpha-helix, estimated from circular dichroism. Removal of the native Zn(II) causes substantial unfolding of the secondary structure. Unlike GAL4(149), the resultant apoprotein is not induced to refold by readdition of Zn(II) at low concentrations.

Amino Acid Sequence↗

PubMed1990

GAL4 transcription factor is not a "zinc finger" but forms a Zn(II)2Cys6 binuclear cluster.

The DNA-binding domain of the transcription factor GAL4, consisting of the 62 N-terminal residues and denoted GAL4(62*), contains a Cys-Xaa2-Cys-Xaa6-Cys-Xaa6-Cys-Xaa2-Cys-Xaa6+ ++-Cys motif, which has been shown previously to bind two Zn(II) or Cd(II) ions. Binding of Zn(II) or Cd(II) is essential for the recognition by GAL4 of the specific palindromic DNA sequence to which it binds upstream of genes for galactose-metabolizing enzymes, the UASG sequence. On the basis of the 113Cd NMR chemical shifts of the two bound 113Cd(II) ions, we propose a binuclear cluster model for this Zn(II)-binding subdomain. 1H-113Cd heteronuclear multiple-quantum NMR spectroscopy and phase-sensitive double-quantum filtered 1H correlation spectroscopy of the 112Cd(II)- and 113Cd(II)-substituted GAL4(62*) derivatives provide direct evidence that the two bound 113Cd(II) ions are coordinated only by the six cysteine residues, two of which form bridging ligands between the two 113Cd(II) ions. The latter can be identified from the pattern of 1H-113Cd J coupling. Thus a binuclear metal ion cluster rather than a "zinc finger" is formed by the six cysteine residues of the GAL4 DNA-binding domain. This model can be directly applied to eight other fungal transcription factors which have been shown to contain similarly spaced Cys6 clusters. 1H NMR spectra of apo-GAL4(62*) suggest conformational fluctuation of the metal-binding subdomain upon removal of Zn(II) or Cd(II). Both Cd(II)2- and Zn(II)2-containing species of GAL4 can be formed, and the similar 1H NMR spectra suggest similar conformations.

Amino Acid Sequence↗

PubMed1989

p10 single-stranded nucleic acid binding protein from murine leukemia virus binds metal ions via the peptide sequence Cys26-X2-Cys29-X4-His34-X4-Cys39.

The RNA binding protein of 56 residues encoded by the extreme 3' region of the gag gene of Rauscher murine leukemia virus (MuLV) has been chemically synthesized by a solid-phase synthesis approach. Since the peptide contains a Cys26-X2-Cys29-X4-His34-X2-Cys39 sequence that is shared by all retroviral gag polyproteins which has been proposed to be a metal binding region, it was of considerable interest to examine the metal binding properties of the complete p10 protein. As postulated, p10 binds the metal ions Cd(II), Co(II), and Zn(II). The Co(II) protein shows a set of d-d absorption bands typical of a tetrahedral Co(II) complex at 695 (epsilon = 565 M-1 cm-1), 642 (epsilon = 655 M-1 cm-1), and 615 nm (epsilon = 510 M-1 cm-1) and two intense bands at 349 (epsilon = 2460 M-1 cm-1) and 314 nm (epsilon = 4240 M-1 cm-1) typical of Co(II)----(-)S- charge transfer. The ultraviolet absorption spectrum also indicates Cd(II) binding by the appearance of a Cd(II)----(-)S- charge-transfer band at 255 nm. The 113Cd NMR spectrum of 113Cd(II)-p10 reveals one signal at delta = 648 ppm. This chemical shift correlates well with that predicted for ligation of 113Cd(II) to three -S- from the three Cys residues of p10. The chemical shift of 113Cd(II)-p10 changes by only 4 ppm upon binding of d(pA)6, indicating that the chelate complex is little changed by oligonucleotide binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗