The major outer membrane protein of Acinetobacter baumannii is the heat-modifiable protein HMP-AB, a porin with a large pore size allowing the penetration of solutes having a molecular weight of up to approximately 800 Da. Cross-linking experiments with glutardialdehyde failed to show any cross-linking between the monomers, a fact that proves again that this porin protein functions as a monomeric porin. The specific activity of this porin was found to be similar to that of other monomeric porins. Tryptic digestion of the outer membrane yielded a 23-kDa fragment of the HMP-AB protein that was resistant to further trypsin treatment. This observation indicates that HMP-AB is assembled in the membrane in a manner similar to monomeric porins. Cloning of the HMP-AB gene revealed an open reading frame of 1038 bp encoding a protein of 346 amino acids and a calculated molecular mass of 35,636 Da. The amino acid sequence and composition were typical of gram-negative bacterial porins: a highly negative hydropathy index, absence of hydrophobic residue stretches, a slightly negative total charge, low instability index, high glycine content, and an absence of cysteine residues. Sequence comparison of HMP-AB with other outer membrane proteins revealed a clear homology with the monomeric outer membrane proteins, outer membrane protein A (OmpA) of Enterobacteria, and outer membrane protein F (OprF) of Pseudomonas sp. Secondary structure analysis indicated that HMP-AB has a 172-amino acid N-terminal domain that spans the outer membrane by eight amphiphilic beta strands and a C-terminal domain that apparently serves as an anchoring protein to the peptidoglycan layer. The results also indicate that HMP-AB belongs to the eight transmembrane beta-strand family of outer membrane proteins.

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Sin1p/Spt2p is a yeast chromatin protein that, when mutated or deleted, alters the transcription of a family of genes presumably by modulating local chromatin structure. In this study, we investigated the ability of different domains of this protein to bind four-way junction DNA (4WJDNA) since 4WJDNA can serve as a model for bent double helical DNA and for the crossed structure formed at the exit and entry of DNA to the nucleosomes. Sequence alignment of Sin1p/Spt2p homologues from 11 different yeast species showed conservation of several domains. We found that three domains of Sin1p/ Spt2p fused to glutathione S-transferase can each bind independently in a structure-specific manner to 4WJDNA as measured in a gel mobility shift assay. A feature common to these domains is a cluster of positively charged amino acids. Modification of this cluster resulted in either abolishment of binding or a change in the binding properties. One of the domains tested clearly bound superhelical DNA, although it failed to induce bending in a circularization assay. Poly-L-lysine, which may be viewed as a cluster of positively charged amino acids, bound 4WJDNA as well. Phenotypic analysis showed that disruption of any of these domains resulted in suppression of a his4-912␦ allele, indicating that each domain has functional significance. We propose that Sin1p/Spt2p is likely to modulate local chromatin structure by binding two strands of double-stranded DNA at their crossover point.Sin1p/Spt2p is a yeast chromatin non-histone protein. While the precise function of the protein is still unknown, it is known to function as a negative transcriptional regulator of a number of genes including SUC2 (1), INO1 (2), and SSA3 (3). Activity of the HO promoter, as measured from an HO promoter driving a lacZ gene (4), is also regulated by SIN1/SPT2. swi1, swi2, and swi3 mutants are unable to transcribe RNA from the HO promoter. However, in sin1/swi double mutants, transcription is restored. Mutants in SPT2 were first identified as suppressors of ty and ␦ insertions in the 5Ј non-coding region of the HIS4 gene (5). As the negative regulation of these genes is overcome by the SW1/SNF chromatin remodeling complex, it was suggested that a function of SIN1p/SPT2p is to somehow maintain chromatin compaction at specific locations in the chromatin.

The yeast chromatin protein Sin1p͞Spt2p has long been studied, but the understanding of its function has remained elusive. The protein has sequence similarity to HMG1, specifically binds crossing DNA structures, and serves as a negative transcriptional regulator of a small family of genes that are activated by the SWI͞SNF chromatin-remodeling complex. Recently, it has been implicated in maintaining the integrity of chromatin during transcription elongation. Here we present experiments whose results indicate that Sin1p͞Spt2 is required for, and is directly involved in, the efficient recruitment of the mRNA cleavage͞polyadenylation complex. This conclusion is based on the following findings: Sin1p͞Spt2 frequently binds specifically downstream of many ORFs but almost always upstream of the first polyadenylation site. It directly interacts with Fir1p, a component of the cleavage͞polyadenylation complex. Disruption of Sin1p͞Spt2p results in foreshortened poly(A) tracts on mRNA. It is synthetically lethal with Cdc73p, which is involved in the recruitment of the complex. This report shows that a chromatin component is involved in 3 end processing of RNA. S in1p is a yeast chromatin nonhistone protein that has beenshown to function as a negative transcriptional regulator of several genes, including Suc2 (1), Ino1 (2), and SSA3 (3). Activity of the HO promoter, as measured from an HO promoter driving a lacZ gene (4), is also affected by Sin1. Sin1 (also known as spt2 in this context) mutants were identified as suppressors of Ty and insertions in the 5Ј noncoding region of the HIS4 gene (5). Because the negative regulation of these genes is overcome by the SW1͞SNF chromatin-remodeling complex (4, 6, 7) and the C-terminal domain of Sin1p interacts with Swi1p (8), it was suggested that a function of SIN1p is to somehow maintain chromatin compaction at specific loci in the chromatin. Peterson et al. (2) found a functional relationship between the C-terminal domain (CTD) of RNA polymerase II and Sin1p, but these data were not pursued further. Sequence analysis of Sin1p showed sequence similarity in two domains to HMG1 (6, 7), a known chromatin protein. Work from our laboratory showed that Sin1p can bind four-way junction and crossing DNA structures (9), supporting the idea that Sin1p binds DNA as it enters and exits the nucleosome.In the context of a global mapping project, Tong et al. (10) reported that there is a synthetic lethal interaction between sin1 and cdc73, a member of the PAF complex. The PAF complex, which accompanies RNA polymerase II during elongation, was shown to have an important function in 3Ј end formation and in polyadenylation (11-13). In addition, a functional interaction was demonstrated between Sin1p and Hpr1p, which is associated with the PAF complex (14).Most recently, evidence has been presented indicating that Sin1p͞Spt2p plays roles in transcription elongation, chromatin structure and genome stability (15). In that study, synthetic lethal interactions were reported between sin1 and paf1, and betwe...

Evidence has recently been mounting suggesting that a number of chromatin components previously thought to primarily or exclusively have structural function, also have a regulatory role in eukaryotic transcription. Notably, in yeast, histone H4 N-terminal sequence has been shown to be required for promoter activation of certain genes in vivo, and mutations in histone H3 (SIN2) or in SIN1 (which has some sequence similarity to HMG1) are able to suppress swi1, swi2, and swi3 mutations, restoring transcription to HO as well as a number of other genes. In this paper we report the identification of a novel protein or protein complex that specifically binds a short sequence in the HO regulatory region on the one hand, and on the other somehow appears to contact the SIN1 protein. We have shown that the DNA binding activity itself does not contain SIN1, since extracts from sin1 delta strains retain the activity. Interestingly, extracts made from cells carrying the dominant sin1-2 point mutation lack the binding activity. Furthermore, bacterially produced sin1-2 protein can dissociate a DNA/protein complex while a similarly produced SIN1 protein has no effect on the complex at similar concentrations. When the DNA sequence to which the protein complex binds is placed in a CYC1 promoter lacking a UAS (upstream activating sequence), it can serve as a weak UAS in a SIN1 dependent way. Our data imply that a sequence specific DNA binding protein(s) may mediate between the SIN1 protein and the basal transcription apparatus transcribing HO.