DNA structure variation at regulatory elements

It is assumed that proteins induce structural changes in DNA. We hypothesize that regulatory DNA sequences assume an active role in protein interaction by adopting structures rendering them distinct from the surrounding DNA. We have studied DNA structure using multidimensional NMR and multiple isotopic labeling. It has been previously observed that there is compression in the major groove at the start site of a T7 viral promoter, and a concomitant widening of the minor groove. To determine if this is also seen in more complex promoters, the bacterial lac and eukaryotic adenovirus major late promoters (AdMLP) were studied. Using 19F NMR, we observed similar results with the lac promoter. We also observed a widening major groove in the AdMLP TATA box corresponding to a bend in the published crystal structure. To obtain structural information on DNA, a protocol for synthesizing 13C/15N DNA was established and used to isotopically label a poly-A tract sequence that is thought to bend in solution. The spectra enabled us to generate a model of the solution structure for this sequence and demonstrate that this sequence is bending in the poly-A tract, in contrast to published crystallographic results. The interaction of the tet repressor with its operator DNA was studied by 19F NMR. Published crystallographic data suggests that two amino acid residues in the DNA binding domain interact directly with the tet operator sequence, inducing a distortion in the DNA. Our results demonstrate that the tet operator displaces a base out of the helical axis in the absence of protein, and that the protein stabilizes this interaction. This suggests a novel interaction between the tet operator and repressor. Additionally, a protocol for the synthesis of 5-fluoro-2′ -deoxycytosine phosphoramidite was devised. This product was used to study an RNA tetraloop sequence that has been reported to form a hairpin structure by NMR and a double helix with an unusual C-U base pair by crystallography. The double-helical form was observed by a 19F-19F coupling under conditions identical to those used in the crystallographic experiments

DNA structure variation at regulatory elements

It is assumed that proteins induce structural changes in DNA. We hypothesize that regulatory DNA sequences assume an active role in protein interaction by adopting structures rendering them distinct from the surrounding DNA. We have studied DNA structure using multidimensional NMR and multiple isotopic labeling. It has been previously observed that there is compression in the major groove at the start site of a T7 viral promoter, and a concomitant widening of the minor groove. To determine if this is also seen in more complex promoters, the bacterial lac and eukaryotic adenovirus major late promoters (AdMLP) were studied. Using 19F NMR, we observed similar results with the lac promoter. We also observed a widening major groove in the AdMLP TATA box corresponding to a bend in the published crystal structure. To obtain structural information on DNA, a protocol for synthesizing 13C/15N DNA was established and used to isotopically label a poly-A tract sequence that is thought to bend in solution. The spectra enabled us to generate a model of the solution structure for this sequence and demonstrate that this sequence is bending in the poly-A tract, in contrast to published crystallographic results. The interaction of the tet repressor with its operator DNA was studied by 19F NMR. Published crystallographic data suggests that two amino acid residues in the DNA binding domain interact directly with the tet operator sequence, inducing a distortion in the DNA. Our results demonstrate that the tet operator displaces a base out of the helical axis in the absence of protein, and that the protein stabilizes this interaction. This suggests a novel interaction between the tet operator and repressor. Additionally, a protocol for the synthesis of 5-fluoro-2′ -deoxycytosine phosphoramidite was devised. This product was used to study an RNA tetraloop sequence that has been reported to form a hairpin structure by NMR and a double helix with an unusual C-U base pair by crystallography. The double-helical form was observed by a 19F-19F coupling under conditions identical to those used in the crystallographic experiments

Aminoacylation of an unusual tRNA(Cys) from an extreme halophile

The extreme halophile Halobacterium species NRC-1 overcomes external near-saturating salt concentrations by accumulating intracellular salts comparable to those of the medium. This raises the fundamental question of how halophiles can maintain the specificity of protein–nucleic acid interactions that are particularly sensitive to high salts in mesophiles. Here we address the specificity of the essential aminoacylation reaction of the halophile, by focusing on molecular recognition of tRNA(Cys) by the cognate cysteinyl-tRNA synthetase. Despite the high salt environments of the aminoacylation reaction, and despite an unusual structure of the tRNA with an exceptionally large dihydrouridine loop, we show that aminoacylation of the tRNA proceeds with a catalytic efficiency similar to that of its mesophilic counterparts. This is manifested by an essentially identical K(m) for tRNA to those of the mesophiles, and by recognition of the same nucleotide determinants that are conserved in evolution. Interestingly, aminoacylation of the halophile tRNA(Cys) is more closely related to that of bacteria than eukarya by placing a strong emphasis on features of the tRNA tertiary core. This suggests an adaptation to the highly negatively charged tRNA sugar-phosphate backbone groups that are the key elements of the tertiary core

Metallothionein: The Sponge Bob of the Cell

Metallothionein is a small protein (approximately 6500 Daltons) found in a variety of cells, but whose function is not completely understood. It contains high amounts of sulfhydryl groups from cysteine which gives it metal binding properties that might play a role in metal detoxification. Furthermore, metallothionein is believed to play an important role in metal metabolism. To understand how metallothionein functions, oligonucleotides that have an overlapping region form a template to be used for a DNA extension reaction. Then amplifying the DNA in Polymerase Chain Reaction (PCR) produces multiple copies that will be used in an in vitro transcription/translation protein synthesis system. This will produce the protein which can be purified and then used to characterize its properties. By studying metallothionein’s ability to bind metals, more can be learned about how the protein functions within the cell. Overall, metallothionein should be studied for its interesting properties which could be useful for studying metal detoxification in the human body

Changes in the ellipticity of NRC-1 CysRS caused by various salts.

<p>The circular dichroism signal of NRC-1 CysRS was measured at 222 nm in different concentrations of group I and group II salts: LiCl (A), NaCl (B), KCl (C), RbCl (D), CsCl (E), MgCl₂ (F), and CaCl₂ (G). Three independent spectra were recorded and the standard deviation for each concentration is reported as error bars. * A consistent signal for 1 M MgCl₂ could not be obtained.</p

Circular Dichroism and Fluorescence Spectroscopy of Cysteinyl-tRNA Synthetase from <i>Halobacterium salinarum</i> ssp. NRC-1 Demonstrates that Group I Cations Are Particularly Effective in Providing Structure and Stability to This Halophilic Protein

<div><p>Proteins from extremophiles have the ability to fold and remain stable in their extreme environment. Here, we investigate the presence of this effect in the cysteinyl-tRNA synthetase from <i>Halobacterium salinarum</i> ssp. NRC-1 (NRC-1), which was used as a model halophilic protein. The effects of salt on the structure and stability of NRC-1 and of <i>E. coli</i> CysRS were investigated through far-UV circular dichroism (CD) spectroscopy, fluorescence spectroscopy, and thermal denaturation melts. The CD of NRC-1 CysRS was examined in different group I and group II chloride salts to examine the effects of the metal ions. Potassium was observed to have the strongest effect on NRC-1 CysRS structure, with the other group I salts having reduced strength. The group II salts had little effect on the protein. This suggests that the halophilic adaptations in this protein are mediated by potassium. CD and fluorescence spectra showed structural changes taking place in NRC-1 CysRS over the concentration range of 0–3 M KCl, while the structure of <i>E. coli</i> CysRS was relatively unaffected. Salt was also shown to increase the thermal stability of NRC-1 CysRS since the melt temperature of the CysRS from NRC-1 was increased in the presence of high salt, whereas the <i>E. coli</i> enzyme showed a decrease. By characterizing these interactions, this study not only explains the stability of halophilic proteins in extremes of salt, but also helps us to understand why and how group I salts stabilize proteins in general.</p></div

Intrinsic fluorescence emission spectra of NRC-1 (red) and <i>E. coli (blue)</i> CysRS.

<p>Fluorescence spectra of NRC-1 and <i>E. coli</i> CysRS using an excitation wavelength of 280 nm in various concentrations of KCl. The solid line represents the spectra collected in 0 M KCl, the dashed line represents the spectra in 500 mM KCl, and the dotted line represents the spectra collected in 3 M KCl.</p

Protein Adaptations in Archaeal Extremophiles

Extremophiles, especially those in Archaea, have a myriad of adaptations that keep their cellular proteins stable and active under the extreme conditions in which they live. Rather than having one basic set of adaptations that works for all environments, Archaea have evolved separate protein features that are customized for each environment. We categorized the Archaea into three general groups to describe what is known about their protein adaptations: thermophilic, psychrophilic, and halophilic. Thermophilic proteins tend to have a prominent hydrophobic core and increased electrostatic interactions to maintain activity at high temperatures. Psychrophilic proteins have a reduced hydrophobic core and a less charged protein surface to maintain flexibility and activity under cold temperatures. Halophilic proteins are characterized by increased negative surface charge due to increased acidic amino acid content and peptide insertions, which compensates for the extreme ionic conditions. While acidophiles, alkaliphiles, and piezophiles are their own class of Archaea, their protein adaptations toward pH and pressure are less discernible. By understanding the protein adaptations used by archaeal extremophiles, we hope to be able to engineer and utilize proteins for industrial, environmental, and biotechnological applications where function in extreme conditions is required for activity