Brigitte Wittmann-Liebold

Micro-sequence analysis of peptides and proteins using 4-NN-dimethylaminoazobenzene 4′-isothiocyanate/phenylisothiocyanate double coupling method

The pheny~sot~ocyanate (Edman) degradation has been a most valuable technique in amino acid sequence determination [I] . One of its inadequacies is, however, the unsatisfactory sensitivity of the identification of PTH-amino acids. This shortcoming has prevented the conventional Edman method from being a practical ~cro-sequencing technique. The combined use of the sensitive dansyl method [2] with the subtractive Edman degradation has largely overcome this problem and contributed, to a great extent, to the sequence analysis of proteins which are available in only limited quantities. Recently, the development of high sensiti~ty sequencing methods has centered on the direct identification of PTHamino acids. These attempts, mostly in conjunction with automated Edman degradations [3,4] include: (1) Use of radioactive Edman reagent 15-71; (2) Incorporation of radioactivity into proteins by in vivo cell culturing in the presence of radioactive amino acids [S] ; (3) Application of high performance liquid chromatography for highly sensitive identification of PTHamino acids [9,10].

Complete nucleotide sequence and gene organization of the broad-host-range plasmid RSF1010

We present the complete nucleotide sequence of RSF1010, a naturally occurring broad-host-range plasmid belonging to the Escherichia coli incompatibility group Q and encoding resistance to streptomycin and sulfonamides. A molecule of RSF1010 DNA consists of 8684 bp and has a G + C content of 61%. Analysis of the distribution of translation start and stop codons in the sequence has revealed the existence of more than 40 open reading frames potentially capable of encoding polypeptides of 60 or more amino acids. To date, products of eleven such potential RSF1010 genes have been identified through the application of controlled expression vector systems, and for eight of them, the reading frame has been confirmed by N- and/or C-terminal amino acid sequence determinations on the purified proteins. The sequencing results are discussed in relation to the systems of replication, host range, conjugal mobilization and antibiotic resistance determinants associated with the RSF1010 plasmid.

The Translational Apparatus

Robin Ray Gutell MCDBiology Campus Box 347 University of Colorado Boulder, Colorado 80309-0347 USA email: rgutell@boulder.colorado.edu The elucidation of 16S and 23S rRNA Higher-Order Structure has been addressed by Comparative Sequence Methods for more than a decade. During these years our comparative methods have evolved as the number of complete 16S and 23S rRNA sequences have increased significantly, resulting in the maturation of the higher-order structure models for 16S and 23S rRNA. With over 1000 16S (and 16S-like) and 200 23S (and 23S-like) sequences at this time, we have strong comparative evidence for the vast majority of all secondary structure base pairings, and are thus quite confident of the majority of the proposed Escherichia coli 16S and 23S rRNA secondary structure. Within the past few years additional rRNA Higher-Order structure constraints have been elucidated; constraints that reveal various RNA structural forms, including lone canonical pairings, pseudoknots, non-canonical pairings, tetra loops, canonical and non-canonical pairings that together forms a parallel (vs. the usual antiparallel) stranded structural element, and suggestive evidence for coaxial stacking of adjacent helices. At this time we question what additional RNA structural constraints can be deciphered with comparative structure methods. To answer such questions, the rRNA sequence collection will need to continue to grow in both number and diversity, and our comparative structure algorithms need to evolve to a more sophisticated level. In an effort to establish the limits for structural similarity, we need to address how different two higher-order structures can be and still be considered analogous. Introductory Statements Since the flrstcomplete 16S (Brosius et al. 1978) and 23S (Brosius et al. 1980) rRNA sequences were determined, comparative analyds of these molecules has progressed in a variety of ways. Maybe foremost for the majority (especially for this audience) is the resulting higher-order structures, which ribosome-ologists utilize to map andlor design their experiments onto. While there is a wealth of information that can and should be elucidated from the sequences that make up the 16S and 23S rRNA datasets, this article will focus on the most obvious and probably experimentally meaningful structural features, namely secondary structure helices, tertiary interactions, and a few interesting examples of other comparatively derived structural constraints. And since much has already been written on comparatively derived rRNA structure [and most recently for an upcoming book on ribosomal RNA (Gutell et al. 1993), this article will only briefly touch on some of the emerging RNA structural features and new The Translational Apparatus, Edited by K.H. Nierhaus et al., Plenum Press, New York, 1993 477 structural possibilities that have been uncovered within the past year or so, leaving the interested reader to investigate elsewhere for a more encompassing perspective of the details of the comparatively derived rRNA structures. Comparative Structure Analysis What is the basis of this method? What might we expect to decipher? This method is rooted in the simple concept that similar or analogous three-dimensional structure can be composed of different primary structures, or in other words many different primary structures can fold into the same isomorphic 3-dimensional structure. Thus natural selection can maintain and act on the higher-order structures of RNA while the primary structure is free to change, although constrained in its divergence. The ribosomal RNA is an ideal molecule to apply such methodology to due to its structural and functional role in the ribosome, and the ribosomes position in protein synthesis and the evolution of the cell (Woese 1980). Underlying this method are a number of key questions that cannot be answered a priori. How much and what types of variance can be tolerated in higher-order structure before these structures are not considered isomorphic? How much overall similarity should we expect to fmd for any RNA molecule? How much overall variance should we expect to find for any RNA molecule (i.e., tRNA: type I vs. type II)? To what extent can these methods identify general folding patterns and to what extent can these methods identify and distinguish subtle and detailed RNA structure (i.e., elucidate the generalized three-dimensional structure for tRNAs; elucidate the detailed features recognized by each of the aminoacyl synthetases for their cognate tRNA)? Will all RNA structural motifs be identified with such methods, or will only a subset of these structural elements be amenable to such methods, i.e., should we expect secondary and all tertiary interactions to be equally decipherable? And lastly, should we anticipate the same overall and/or detailed structural (and even biological) constraints within phylogenetic ally related vs. distant structures? A quick glimpse at the progression of our rRNA structure models Although these questions are not (yet) answerable, the comparative analysis of the rRNAs (and all RNAs for that matter) has advanced in stages, in part so the results from each stage with their underlying assumptions can be evalulated before moving on to the next stage, and in part due to the significant increase in the number of sequences, development of the underlying correlation analysis algorithms, and the fact that we believe there is more structural detail to be found at the completion of each stage. This analysis started with basic assumptions that were congruent with principles elucidated with experimental methods. Initially the comparative structure searched for the helices that compose the overall secondary structure [For 16S rRNA: (Woese et al. 1980, Stiegler et al. 1980, Zwieb et al. 1981), and 23S rRNA: (Nolier et al. 1981, Glotz et al. 1981, and Branlant et al. 1981) rRNAs]. These methods specifically searched for canonical base pairings (ie. A-U and G-C) arranged contiguously and in an antiparallel orientation. These structures were tested and evaluated with each new rRNA sequence, resulting in numerous refmements in the secondary structures [Many references not noted here]. The specific search for helical elements gave way to a more generalized, non-structure based method. This method (Gutell et al. 1985) transformed the pattern of nucleotides at each column in the sequence alignment to a number pattern, which was based on the pattern of conservation and variance at every position in the molecule. Similar number patterns were subsequently grouped and analyzed, resulting in refmements in the secondary structure, and several proposed tertiary interactions (Gutell et al. 1985, 1986). Equally significant this simple algorithm uncovered a few basic principles of RNA structure, namely canonical pairings, and contiguous and antiparallel arrangement of such pairings [It should be noted that this method searched for columns (nucleotide positions) with similar patterns of variation or covariance, regardless of the nucleotide and pairing types. It so happened that the underlying pairs

A device coupled to a modified sequenator for the automated conversion of anilinothiazolinones into PTH amino acids

Abstract A device for the automated conversion of anilinothiazolinones of amino acids to their phenylthiohydantoin derivatives has been incorporated into a modifled Beckman sequenator. The construction of the device and its cyclic operating principle are described, and a program for its operation is given. The automated conversion procedure has been used in the sequenator for 4 years, and its significant advantages over manual conversion with respect to the improved yields and purity of phenylthiohydantoin amino acids are discussed.

MALDI-MS for C-terminal sequence determination of peptides and proteins degraded by carboxypeptidase Y and P

Matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS) has been used for C‐terminal amino acid sequence determination of peptides and proteins. The usefulness of MALDI‐MS was demonstrated by analyzing peptide mixtures (C‐terminal peptide ladder) which were generated by enzymatic digestion of substance P, glucagon, angiotensinogen, insulin B chain and myoglobin with the exopeptidases carboxypeptidase Y and P. The results clearly show that up to 11 amino acid residues can be determined in the pmol range by analyzing the molecular masses of the truncated peptides. For proteins it is possible to investigate enzymatic or chemical digests in the same manner.

Isolation, characterization and microsequence analysis of a small basic methylated DNA-binding protein from the Archaebacterium, Sulfolobus solfataricus

DNA-binding proteins have been extracted from the thermoacidophilic archaebacterium Sulfolobus solfataricus strain P1, grown at 86 degrees C and pH 4.5. These proteins, which may have a histone-like function, were isolated and purified under standard, non-denaturing conditions, and can be grouped into three molecular mass classes of 7, 8 and 10 kDa. We have purified to homogenity the main 7 kDa protein and determined its DNA-binding affinity by filter binding assays and electron microscopy. The Stokes radius of gyration indicates that the protein occurs as a monomer. The complete amino-acid sequence of this protein contains 14 lysine residues out of 63 amino acids and the calculated Mr is 7149. Five of the lysine residues are partially monomethylated to varying extents and the methylated residues are located exclusively in the N-terminal (positions 4 and 6) and the C-terminal (positions 60, 62 and 63) regions only. The protein is strongly homologous to the 7 kDa proteins of Sulfolobus acidocaldarius with the highest homology to protein 7d. Accordingly, the name of this protein from S. solfataricus was assigned as DNA-binding protein Sso7d.

Purification and characterization of ten new rice NaCl-soluble proteins: identification of four protein-synthesis inhibitors and two immunoglobulin-binding proteins.

Ten new proteins from rice (Oryza saliva L. cv. Bahia) including four protein-synthesis inhibitors and two immunoglobulin E (IgE)-binding proteins have been isolated and characterized. These proteins as well as one previously known component, α-globulin, were purified from a 0.5 M NaCl extract of rice endosperm by a new, apparently non-denaturing, isolation procedure developed for rice proteins. The method is based on extractions of this complex protein mixture with a diluted volatile salt solution and an aqueous solution of ethanol. This preliminary step results in an improvement in the separation of these proteins, thus facilitating their subsequent purification by reversed-phased high-performance liquid chromatography. These new proteins have similar relative molecular masses (Mrs) from 11000 to 17000. The purity of the proteins was analyzed by micro two-dimensional gel electrophoresis. Four of these components were found to be in-vitro protein-synthesis inhibitors in a cell-free system from rat brain. The NH2-terminal amino-acid sequences of these four inhibitors were determined from 12 to 26 cycles after direct blotting of the separated proteins from electrophoresis gels. Three of these proteins with Mrs between 16000 and 17000 showed a high degree of homology ranging from 57% to 75% but seem to be unrelated to the fourth inhibitor. In addition, the α-globulin and one of the new low-molecular-weight proteins of Mr 12500 seemed to show allergenic properties since they bound IgE antibodies from the sera of hypersensitive patients. Boths proteins have blocked NH2-terminal amino acids.

Coat proteins of strains of two RNA viruses: Comparison of their amino acid sequences

SummaryThe amino acid sequences of four strains of tobacco mosaic virus isolated in different parts of the world are compared. The differences between the strains are discussed with respect to special proteinchemical features (such as beginning of the chain, deletion of amino acids, number of different amino acids, sizes and distribution of regions with invariable amino acids) and with respect to the possibility of deducing the most probable nucleotide sequence for the coat protein cistron of tobacco mosaic virus.The complete amino acid sequences of the two RNA bacteriophage strains fr and f2 are compared. According to their coat proteins three groups of phages can be formed: 1) MS 2, f2 M 12 and R 17, 2) fr and 3) Qβ.

The primary structure of L11, the most heavily methylated protein from Escherichia coli ribosomes

Protein L11 from the large ribosomal subunit of E. colt was reported to be part of the peptidyltransferase centre [1 -4] . However, a 50 S derived core lacking L11 was described to be fully active in peptidyltransferase activity [5,6] and L16 was found to be essential for this activity [7]. Recently, this controversy was settled by showing that L11 is important for the assembly of L16 when all the components are present in stoichiometric amounts. The importance of L11 can be reduced by a ten-fold excess of L16 [81. Protein L11, which binds specifically to 23 S ribosomal RNA when prepared under non-denaturing conditions [9], can be cross-linked to proteins L7/12 and L10 [10,I 1]. L11 has been identified by photoaffinity labelling as one of the proteins involved in EF-G-dependent GDP binding [12]. Immune electron microscopy revealed two antibody binding sites for L11 on the 50 S subunit and therefore indicates a slightly elongated shape for this protein [13]. This conclusion is in agreement with physical studies on the isolated protein resulting in an axial ratio of 5-6:1 [ 14]. Several mutants with altered L11 have been isolated [15-17] and two of them have a relaxed control of RNA synthesis [18]. This indicates that L11 is a participant in the synthesis of ppGpp and pppGpp. Protein L11 is the most extensively methylated of the E. colt ribosomal proteins. The methyl groups were found in one residue of lysine as Ne-trimethyllysine, and in one unidentified neutral amino acid [19,20]. Recently, N-trimethylalanine was identifled in the mixture of 50 S proteins and was assumed to be the unidentified neutral amino acid in L11 [21]. Furthermore, two other amino acids,Ne-monomethyllysine and dimethylarginine, were reported to be also present in L11 [17,20,22]. This paper summarizes the determination of the complete amino acid sequence of protein L11 including the nature, quantity and location of the different methylated derivatives. Protein LI 1 consists of 141 amino acids and the blocked N-terminal residue was shown to be N-trimethylalanine. Furthermore, two Ne-trimethyllysines were found in this protein. No other methylated amino acid residues could be found in the sequence determination of protein L11.

Ribosomal proteins: XXII. Studies on the altered protein S5 from a spectinomycin-resistant mutant of Escherichia coli☆

Abstract Reconstitution tests confirmed that ribosomal protein S5 from 30 s subunits is responsible for resistance of ribosomes to the antibiotic spectinomycin. Protein S5 from resistant ribosomes was isolated and digested with trypsin. The trypsin peptides were separated by column and paper chromatography and their amino acid compositions determined. Comparison of the peptides from the resistant mutant and the wild type revealed an amino acid exchange in peptide T10. Sequence analyses demonstrated that a serine residue in peptide T10 from wild type was replaced by proline in peptide T10 of the resistant mutant.