Laser-induced dissociation of phosphorylated peptides using matrix assisted laser desorption/ionization tandem time-of-flight mass spectrometry

Reversible phosphorylation is one of the most important posttranslational modifications of cellular proteins. Mass spectrometry is a widely used technique in the characterization of phosphorylated proteins and peptides. Similar to nonmodified peptides, sequence information for phosphopeptides digested from proteins can be obtained by tandem mass analysis using either electrospray ionization or matrix assisted laser desorption/ionization (MALDI) mass spectrometry. However, the facile loss of neutral phosphoric acid (H₃PO₄) or HPO₃ from precursor ions and fragment ions hampers the precise determination of phosphorylation site, particularly if more than one potential phosphorylation site or concensus sequence is present in a given tryptic peptide. Here, we investigated the fragmentation of phosphorylated peptides under laser-induced dissociation (LID) using a MALDI-time-of-flight mass spectrometer with a curved-field reflectron. Our data demonstrated that intact fragments bearing phosphorylated residues were produced from all tested peptides that contain at least one and up to four phosphorylation sites at serine, threonine, or tyrosine residues. In addition, the LID of phosphopeptides derivatized by N-terminal sulfonation yields simplified MS/MS spectra, suggesting the combination of these two types of spectra could provide an effective approach to the characterization of proteins modified by phosphorylation.     

Transcription of polyoma virus DNA after interaction with nuclear proteins in vitro.

Analysis of the nucleotide sequence at the 5′-triphosphate termini of RNA chains synthesized by T7 RNA polymerase from T7 DNA template indicates that nearly all RNA chains synthesized in this polymerase reaction contain the sequence, pppGpGp. In addition, studies carried out on T7 DNA-dependent ³²PP_i exchange into ribonucleoside triphosphates suggest that immediately following the guanine residues at the 5′-end of RNA formed in the T7 RNA polymerase reaction, there is one or more adenine residues. These results indicate a high degree of specificity of initiation of RNA synthesis by T7 RNA polymerase.     

Interaction of R17 Coat Protein With Its RNA Binding Site For Translational Repression

Abstract

The interaction between bacteriophage R17 coat protein and its RNA binding site for translational repression was studied as an example of a sequence-specific RNA-protein interaction. A nitrocellulose filter retention assay is used to demonstrate equimolar binding between the coat protein and a synthetic 21 nucleotide RNA fragment. The Kj at 2°C in a buffer containing 0.19 M salt is about 1 nM. The relatively weak ionic strength dependence of K_a and a ΔH = ?19 kcal/mole indicates that most of the binding free energy is due to non-electrostatic interactions. Since a variety of RN As failed to compete with the 21 nucleotide fragment for coat protein binding, the interaction appears highly sequence specific.

We have synthesized more than 30 different variants of the binding site sequence in order to identify the portions of the RNA molecule which are important for protein binding. Out of the five single stranded residues examined, four were essential for protein binding whereas the fifth could be replaced by any nucleotide. One variant was found to bind better than the wild type sequence. Substitution of nucleotides which disrupted the secondary structure of the binding fragment resulted in very poor binding to the protein. These data indicated that there are several points of contact between the RNA and the protein and the correct hairpin secondary structure of the RNA is essential for protein binding.     

Guidelines for the selection of highly effective siRNA sequences for mammalian and chick RNA interference

In the present study, the relationship between short interfering RNA (siRNA) sequence and RNA interference (RNAi) effect was extensively analyzed using 62 targets of four exogenous and two endogenous genes and three mammalian and Drosophila cells. We present the rules that may govern siRNA sequence preference and in accordance with which highly effective siRNAs essential for systematic mammalian functional genomics can be readily designed. These rules indicate that siRNAs which simultaneously satisfy all four of the following sequence conditions are capable of inducing highly effective gene silencing in mammalian cells: (i) A/U at the 5′ end of the antisense strand; (ii) G/C at the 5′ end of the sense strand; (iii) at least five A/U residues in the 5′ terminal one-third of the antisense strand; and (iv) the absence of any GC stretch of more than 9 nt in length. siRNAs opposite in features with respect to the first three conditions give rise to little or no gene silencing in mammalian cells. Essentially the same rules for siRNA sequence preference were found applicable to DNA-based RNAi in mammalian cells and in ovo RNAi using chick embryos. In contrast to mammalian and chick cells, little siRNA sequence preference could be detected in Drosophila in vivo RNAi.     

Sequencing a protein by x-ray crystallography. II. Refinement of yeast hexokinase B co-ordinates and sequence at 2.1 A resolution.

Although the amino acid sequence of yeast hexokinase B has not been determined by chemical means, crystallographic refinement of the hexokinase monomer was carried out at 2.1 Å resolution to improve both the atomic co-ordinates and the amino acid sequence, which had been obtained from a 2.5 Å electron density map. The atomic co-ordinates were adjusted by real-space refinement into a multiple isomorphous replacement map, followed by automated difference Fourier refinement, and restrained parameter structure factor least-squares refinement. The amino acid sequence was altered periodically after visual inspection of (F_o ? F_c) difference electron density maps. Evidence of the improvement in the amino acid sequence was provided by the better agreement between the X-ray and chemically derived amino acid compositions, and most importantly by the ability to locate two short peptides which had been chemically sequenced. While only 6 out of the 18 residues in these two peptides agree with the sequence of the original model, 12 residues agree with the sequence of the refined model and the others differ by only an atom or two. The refined model contains 3293 of of the 3596 non-hydrogen atoms expected from the amino acid composition and 152 bound water molecules. The crystallographic R factor at 2.1 Å is 0.25.We show that there are several advantages to refining the structure of even a protein of unknown sequence. (1) Improved phases can be obtained to the resolution limit of the diffraction pattern starting with a model derived from a 2.5 Å map. (2) The accuracy of the amino acid sequence derived by X-ray methods alone can be substantially improved. (3) Functionally important residues can be identified before chemical sequence information is available. (4) The improved X-ray sequence should greatly reduce the effort required to obtain a chemical sequence; since peptides as short as eight or nine residues can be located in the refined X-ray sequence, peptides do not need to be overlapped by chemical means.     

Unique nucleotide sequence adjacent to the poly (A) region in yeast RNA

Yeast cells growing in a low phosphate medium were labeled with a pulse of ³²P_i or [³H]adenine and harvested after 15 minutes. Total RNA was extracted and digested with ribonuclease T₁. Poly(A)-rich fragments were isolated from the digest by hybridization to poly(U) impregnated fiberglass filters. Gel filtration showed the fragments to have a uniform chain length of about sixteen. Analysis of the composition gave (A₁₁, C₄, U). Complete pancreatic ribonuclease and partial spleen phosphodiesterase digests gave the sequence of the 5′ end of the fragment as CpApApUp-. Since the fragment was a ribonuclease T₁ product, the data points to a unique sequence of at least five residues, -GpCpApApUp-, adjacent to the poly(A)-rich terminus of pulse-labeled yeast mRNA. The remainder of the poly(A)-rich fragment consists of A residues with a few randomly interspaced C residues. The known specificity of yeast poly(A) polymerase can account for the presence of C residues in poly(A) tracts.     

Isolation and characterization of two wound-regulated tomato extensin genes

Extensins comprise a family of structural cell wall hydroxyproline-rich glycoproteins in plants. Two tomato genomic clones, Tom J-10 and Tom L-4, were isolated from a tomato genomic DNA library byin situ plaque hybridization with extensin DNA probes. Tom J-10 encoded an extensin with 388 amino acid residues and a predicted molecular mass of 43 kDa. The Tom J-10 encoded extensin lacked a typical signal peptide sequence, but contained two distinct protein domains consisting of 19 tandem repeats of Ser-Pro₄-Ser-Pro-Lys-Tyr-Val-Tyr-Lys at the amino terminus which were directly followed by 8 tandem repeats of the consensus sequence Ser-Pro₄-Tyr₃-Lys-Ser-Pro₄-Ser-Pro at the carboxy terminus. RNA blot hybridization analysis with the Tom J-10 extensin probe demonstrated the presence of a 4.0 kb tomato stem mRNA which accumulated markedly in response to wounding. Tom L-4 encoded an extensin with 322 amino acid residues and a predicted molecular mass of 35 kDa. The Tom L-4 encoded extensin contained a typical signal peptide sequence at the amino terminus and was followed by at least 3 distinct domains. These domains consisted of an amino terminal domain containing several Lys-Pro and Ser-Pro₄ repeat units, a central domain with repeats of the consensus sequence Ser-Pro_2–5-Thr-Pro-Ser-Tyr-Glu-His-Pro-Lys-Thr-Pro, and a carboxy terminal domain containing repeats of the consensus sequence Ser-Ser-Pro₄-Ser-Pro-Ser-Pro₄-Thr-Tyr_1–3. RNA blot hybridization analysis with the Tom L-4 extensin probe demonstrated the presence of a 2.6 kb tomato stem mRNA which accumulated in response to wounding.     

Specificity and Evolutionary Conservation of the Escherichia coli RNA Pyrophosphohydrolase RppH

Bacterial RNA degradation often begins with conversion of the 5′-terminal triphosphate to a monophosphate by the RNA pyrophosphohydrolase RppH, an event that triggers rapid ribonucleolytic attack. Besides its role as the master regulator of 5′-end-dependent mRNA decay, RppH is important for the ability of pathogenic bacteria to invade host cells, yet little is known about how it chooses its targets. Here, we show that Escherichia coli RppH (EcRppH) requires at least two unpaired nucleotides at the RNA 5′ end and prefers three or more such nucleotides. It can tolerate any nucleotide at the first three positions but has a modest preference for A at the 5′ terminus and either a G or A at the second position. Mutational analysis has identified EcRppH residues crucial for substrate recognition or catalysis. The promiscuity of EcRppH differentiates it from its Bacillus subtilis counterpart, which has a strict RNA sequence requirement. EcRppH orthologs likely to share its relaxed sequence specificity are widespread in all classes of Proteobacteria, except Deltaproteobacteria, and in flowering plants. By contrast, the phylogenetic range of recognizable B. subtilis RppH orthologs appears to be restricted to the order Bacillales. These findings help to explain the selective influence of RppH on bacterial mRNA decay and show that RppH-dependent degradation has diversified significantly during the course of evolution.     

Fingerprinting the junctions of RNA structure by an open-paddlewheel diruthenium compound

RNA function is determined by its structural organization. The RNA structure consists of the combination of distinct secondary structure motifs connected by junctions that play an essential role in RNA folding. Selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) probing is an established methodology to analyze the secondary structure of long RNA molecules in solution, which provides accurate data about unpaired nucleotides. However, the residues located at the junctions of RNA structures usually remain undetected. Here we report an RNA probing method based on the use of a novel open-paddlewheel diruthenium (OPW-Ru) compound [Ru₂Cl₂(µ-DPhF)₃(DMSO)] (DPhF = N,N′-diphenylformamidinate). This compound has four potential coordination sites in a singular disposition to establish covalent bonds with substrates. As a proof of concept, we have analyzed the reactivity of OPW-Ru toward RNA using two viral internal ribosome entry site (IRES) elements whose function depends on the structural organization of the molecule. Our study suggests that the compound OPW-Ru preferentially attacks at positions located one or two nucleotides away from junctions or bulges of the RNA structure. The OPW-Ru fingerprinting data differ from that obtained by other chemical reagents and provides new information about RNA structure features.     

Nucleotide sequence of cDNA clones encoding the complete precursor for subunit delta of thylakoid-located ATP synthase from spinach

The nucleotide sequence of the entire nuclear-encoded precursor for subunit delta of the ATP synthase from spinach thylakoid membranes was determined by cDNA sequencing. Appropriate recombinant DNAs were selected from pBR322 and lambda gt11 libraries made from polyadenylated RNA of greening spinach seedlings. The mature protein consists of 187 amino acid residues corresponding to a molecular weight of 20468. The precursor protein (257 amino acid residues; M _r=27676) is probably processed between a Met-Val bond. The predicted secondary structure of the transit sequence (70 residues; 7.2 kDa) resembles that of the Rieske Fe/S polypeptide, but shows little similarity with those of stromal or luminal proteins. The comparison of the chloroplast delta amino acid sequence with the published delta sequences from respiratory ATP synthases of bacterial and mitochondrial sources and from the thylakoid ATP synthase of the cyanobacterium Synechococcus suggests substantial divergence at the genic level although structural elements appear to be remarkably conserved.     

Crotalus adamanteus phospholipase A2-α: Subunit structure,NH2-terminal sequence,and homology with other phospholipases

Automated Edman degradation of reduced and carboxymethylated phospholipase A₂-α from Crotalus adamanteus venom revealed a single amino acid sequence extending 30 residues into the protein from the amino terminus. The singularity of the sequence and the yields of the phenylthiohydantoin amino acids thus obtained indicate that the subunits comprising the phospholipase dimer are identical. Further chemical evidence in support of subunit identity was obtained by cleavage of phospholipase A₂-α with cyanogen bromide. Compositional analysis of the protein revealed one residue of methionine per monomer and the sequence determination placed this amino acid at position 10 in the sequence of 133 amino acids. Cyanogen bromide cleavage of the protein, followed by reduction and carboxymethylation afforded the expected 2 fragments: an NH₂-terminal decapeptide (CNBr-1) and a larger COOH-terminal fragment of 123 residues (CNBr-II). Automated Edman degradation of the latter has extended the sequence analysis to 54 residues in the NH₂-terminal segment of the monomer chain. Comparison of this sequence with those derived for phospholipases from other snake venoms, from bee venom, and from porcine pancreas has revealed striking homologies in this region of the molecules. As expected on the basis of their phylogenetic classification, the phospholipases from the pit vipers C. adamanteus and Agkistrodon halys blomhoffii are more similar to one another in sequence than to the enzyme from the more distantly related viper, Bitis gabonica. Furthermore, the very close similarities in sequence observed among all of these phospholipases in regions corresponding to residues 24 through 53 in the C. adamanteus enzyme suggest that this segment of the polypeptide plays an important role in phospholipase function and probably constitutes part of the active site.     

Bacterial genotyping by 16S rRNA mass cataloging

Background  

It has recently been demonstrated that organism identifications can be recovered from mass spectra using various methods including base-specific fragmentation of nucleic acids. Because mass spectrometry is extremely rapid and widely available such techniques offer significant advantages in some applications. A key element in favor of mass spectrometric analysis of RNA fragmentation patterns is that a reference database for analysis of the results can be generated from sequence information. In contrast to hybridization approaches, the genetic affinity of any unknown isolate can in principle be determined within the context of all previously sequenced 16S rRNAs without prior knowledge of what the organism is. In contrast to the original RNase T₁ cataloging method, when digestion products are analyzed by mass spectrometry, products with the same base composition cannot be distinguished. Hence, it is possible that organisms that are not closely related (having different underlying sequences) might be falsely identified by mass spectral coincidence. We present a convenient spectral coincidence function for expressing the degree of similarity (or distance) between any two mass-spectra. Trees constructed using this function are consistent with those produced by direct comparison of primary sequences, demonstrating that the inherent degeneracy in mass spectrometric analysis of RNA fragments does not preclude correct organism identification.     

Cys-92, Cys-95, and the C-terminal 12 residues of the <Emphasis Type="Italic">Vibrio harveyi</Emphasis> ferric uptake regulator (Fur) are functionally inessential

Ferric uptake regulator (Fur) is a global regulator involved in multiple aspects of bacterial life. The gene encoding the Vibrio harveyi Fur (Fur_vh) was cloned from a pathogenic V. harveyi strain isolated from diseased fish. Furvh shares 77% overall sequence identity with the Escherichia coli Fur (Fur_Ec) and could complement a mutant of Fur_Ec. Like Fur_Ec, Fur_Vh, possesses two cysteine residues at positions 92 and 95, yet unlike Fur_Ec, in which these cysteine residues constitute part of the metal ion coordination site and hence are vital to the repressor activity, C92 and C95 of Fur_Vh proved to be functionally inessential. Further study identified a Vibrio Fur signature sequence, which is preserved in all the ten Vibrio Fur proteins that have been discovered to date but in none of the non-vibrio Fur proteins. Site-directed and random mutation analyses of the signature residues, the cysteine residues, and seven highly charged amino acid residues indicated that D9, H32, C137, and K138 of Fur_vh are functionally important but D9, C137, and K138 can be replaced by more than one functional substitutes. Systematic deletion analysis demonstrated that the C-terminal 12 residues of Fur_Vh are functionally inessential. These results (i) indicated that the activation mechanism, or certain aspects of which, of Fur_Vh is possibly different from that of Fur_Ec; and (ii) suggested that it is not very likely that the C-terminal 12 residues play any significant role in the activation or stability of Fur_Vh; and (iii) provided insights into the potential function of the local structure involving C137 and K138.     

N(omega)-arginine dimethylation modulates the interaction between a Gly/Arg-rich peptide from human nucleolin and nucleic acids

We studied the interaction between a synthetic peptide (sequence Ac-GXGGFGGXGGFXGGXGG-NH₂, where X = arginine, N^ω,N^ω-dimethylarginine, DMA, or lysine) corresponding to residues 676–692 of human nucleolin and several DNA and RNA substrates using double filter binding, melting curve analysis and circular dichroism spectroscopy. We found that despite the reduced capability of DMA in forming hydrogen bonds, N^ω,N^ω-dimethylation does not affect the strength of the binding to nucleic acids nor does it have any effect on stabilization of a double-stranded DNA substrate. However, circular dichroism studies show that unmethylated peptide can perturb the helical structure, especially in RNA, to a much larger extent than the DMA peptide.     

Identification of amino acid residues essential for reconstitutive activity of subunit IV of the cytochrome bc1 complex from Rhodobacter sphaeroides

A region of subunit IV comprising residues 77-85 is identified as essential for interaction with the core complex to restore the bc₁ activity (reconstitutive activity). Recombinant subunit IV mutants with single or multiple alanine substitution at this region were generated and characterized to identify the essential amino acid residues. Residues 81-84, with sequence of YRYR, are required for reconstitutive activity of subunit IV, because a mutant with these four residues substituted with alanine has little activity, while a mutant with alanine substitution at residues 77-80 and 85 have the same reconstitutive activity as that of the wild-type IV. The positively charged group at R-82 and R-84 and both the hydroxyl group and aromatic group at Y-81 and Y-83 are essential. The interactions between these four residues of subunit IV and residues of core subunits are also responsible for the stability of the complex. However, these interactions are not essential for the incorporation of subunit IV into the bc₁ complex.     

The complete amino acid sequence of ribosomal protein S12 from Bacillus stearothermophilus

The amino acid sequence of ribosomal protein S12 from Bacillus stearothermophilus has been completely determined. The sequence data were mainly obtained by manual sequencing of peptides derived from digestion with trypsin, Staphylococcus aureas protease and pepsin. A few overlaps of tryptic peptides were established by DNA sequence analysis of a chromosomal fragment containing the rpsL gene coding for ribosomal protein S12. The protein contains 138 amino acid residues and has an M_r of 15208. Comparison of this sequence with the sequences of the ribosomal S12 proteins from E. coli as well as from Euglena, tobacco and liverwort chloroplasts shows that 75% of the amino acid residues are identical within the S12 proteins of all four species. Therefore, S12 is the most strongly conserved ribosomal protein known so far.