C-terminal Flanking Sequences Research Articles

DNA Bending by the Chromosomal Protein HMG1 and Its High Mobility Group Box Domains: EFFECT OF FLANKING SEQUENCES

Abstract HMG1 is an evolutionarily highly conserved chromosomal protein consisting of two folded DNA-binding domains, A and B (“high mobility group (HMG) boxes”), and an acidic C-terminal domain. Several lines of evidence suggest that previously reported sequence-independent DNA bending and looping by HMG1 and its HMG box domains might be important for the proposed role of the protein in transcription and recombination. We have used ligase-mediated circularization assays to investigate the contribution of the individual A and B HMG1 box domains and of the linker region between A/B- and B/C-domains, which flank the “minimal” B-domain (residues 92–162), to the ability of the HMG1 protein (residues 1–215) to bend DNA. Neither the minimal B-domain nor the minimal B-domain with a 7-residue N-terminal extension (85TKKKFKD91) bent the DNA. The attachment of an extra 18-residue C-terminal additional extension (residues 163–180) to the minimal B-domain had only a small effect on the ability of the HMG box to bend DNA. On the other hand, circularization assay with a B-domain having both 7-residue N-terminal and 18-residue C-terminal flanking sequences (residues 85–180) revealed a strong bending of the DNA, suggesting that both extensions are a prerequisite for efficient DNA bending by the B-domain. We have also shown that a single lysine residue (Lys90) in a short N-terminal sequence 90KD91 attached to the B-domain is sufficient for strong distortion of DNA by bending, provided that the B-domain is flanked by the 18-residue C-terminal flanking sequence. Although the DNA bending potential of HMG1 seems to be predominantly due to the B-domain flanked by basic sequences, covalent attachment of the A- and B-domains is necessary for efficient DNA flexure and the ability of the (A+B)-bidomain to bend DNA is further modulated in the native HMG1 protein by its acidic C-domain.

Identification, expression, and crystallization of the protease-resistant conserved domain of synapsin I.

A 35-37-kDa protease-resistant domain of synapsin Ia/ Ib, apparently produced by low levels of endogenous proteases in vapor diffusion droplets, slowly formed crystals diffracting X-rays to approximately 10 A resolution. The fragment mainly consisted of the highly conserved C domain common to the synapsin I/II family plus short N- and C-terminal flanking segments. Two constructs (SynA and SynB) of synthetic gene fragments coding for the C domain of synapsin with or without C-terminal flanking sequence were expressed in Escherichia coli as fusion proteins attached to the soluble protein glutathione-S-transferase. The fusion proteins were purified by affinity chromatography. Subsequent in situ cleavage with TEV protease resulted in the release of highly pure synapsin fragments, which were further purified by ion exchange chromatography. SynA and SynB formed crystals within three days, which diffracted to better than 3 A using a conventional X-ray source and to about 2 A using a synchrotron X-ray source. SynA crystals have the symmetry of the trigonal space groups P3(1)21 or P3(2)21 and the unit cell dimensions a = b = 77.4 A, c = 188.5 A, alpha = beta = 90 degrees, gamma = 120 degrees. SynB crystals have the symmetry of the orthorhombic space group C222(1) with the unit cell dimension a = 104.6 A, b = 113.3 A, and c = 273.8 A.

Fine specificity of the B-cell epitopes recognized in HIV-1 NEF by human sera

We have previously used partially overlapping synthetic nonapeptides to characterize the human natural antibody response against HIV-1 negative regulatory factor (NEF), and identified nine 5 to 13 amino acid long regions that were recognized by sera of HIV-1-infected individuals. In this report we define the minimal size of these epitopes with the use of shorter, from 3 to 8 amino acid long partially overlapping peptides covering the complete sequence of the previously identified reacting regions and the N- and C-terminal flanking sequences. We also introduce a new method for the analysis of the reactivities obtained with peptides of different lengths. In six of the antigenic regions the epitopes were found to be noncontiguous and to consist of multiple, down to three amino acid long separate reactive stretches (epitope 1: WSK, VGW, TVRERMRR; epitope 3A: PLRPM, SHFLK; epitope 313: SQRRQD, DLW; epitope 3C: IYHT, QGYFPDWQN; epitope 4: SLL, VSL; epitope 5: EVLEWRFDSR, VAR). Three epitopes were clearly linear (epitope 2: CAWLE; epitope 3D: LTFGWC; epitope 6: PEYF). Interestingly, five of the minimized B-cell epitopes (1, 3A, 3C, 3D, 5) recognized by human sera overlap totally or partly with the previously identified T-cell epitopes in HIV-1 NEF. Also, only three of the epitopes (3C, 3D, 5) were in a computer-based homology search shown to contain strictly NEF-specific sequences.

Sequence analysis of the Clostridium stercorarium celZ gene encoding a thermoactive cellulase (Avicelase I): Identification of catalytic and cellulose-binding domains

The nucleotide sequence of the celZ gene coding for a thermostable endo-beta-1,4-glucanase (Avicelase I) of Clostridium stercorarium was determined. The structural gene consists of an open reading frame of 2958 bp which encodes a preprotein of 986 amino acids with an Mr of 109,000. The signal peptide cleavage site was identified by comparison with the N-terminal amino acid sequence of Avicelase I purified from C. stercorarium culture supernatants. The recombinant protein expressed in Escherichia coli is proteolytically cleaved into catalytic and cellulose-binding fragments of about 50 kDa each. Sequence comparison revealed that the N-terminal half of Avicelase I is closely related to avocado (Persea americana) cellulase. Homology is also observed with Clostridium thermocellum endoglucanase D and Pseudomonas fluorescens cellulase. The cellulose-binding region was located in the C-terminal half of Avicelase I. It consists of a reiterated domain of 88 amino acids flanked by a repeated sequence about 140 amino acids in length. The C-terminal flanking sequence is highly homologous to the non-catalytic domain of Bacillus subtilis endoglucanase and Caldocellum saccharolyticum endoglucanase B. It is proposed that the enhanced cellulolytic activity of Avicelase I is due to the presence of multiple cellulose-binding sites.