The RepE initiator is a double-stranded and single-stranded DNA-binding protein that forms an atypical open complex at the onset of replication of plasmid pAMbeta 1 from Gram-positive bacteria.
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The RepE protein of the broad host range pAMbeta1 plasmid from Gram-positive bacteria is absolutely required for replication. To elucidate its role, we purified the protein to near homogeneity and analyzed its interactions with different nucleic acids using gel retardation assays and footprinting experiments. We show that RepE is monomeric in solution and binds specifically, rapidly, and durably to the origin at a unique double-stranded binding site immediately upstream from the initiation site of DNA replication. The binding induces only a weak bend (31 degrees ). Unexpectedly, RepE also binds nonspecifically to single-stranded DNA with a 2-4-fold greater affinity than for double-stranded origin. On a supercoiled plasmid, RepE binding to the double-stranded origin leads to the denaturation of the AT-rich sequence immediately downstream from the binding site to form an open complex. This open complex is atypical since (i) its formation requires neither multiple RepE binding sites on the double-stranded origin nor strong bending of the origin, (ii) it occurs in the absence of any cofactors (only RepE and supercoiling are required), and (iii) its melted region serves as a substrate for RepE binding. These original properties together with the fact that pAMbeta1 replication depends on a transcription step through the origin on DNA polymerase I to initiate replication and on a primosome to load the replisome suggest that the main function of RepE is to assist primer generation at the origin. (+info)
Tetraplex formation by the progressive myoclonus epilepsy type-1 repeat: implications for instability in the repeat expansion diseases.
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The repeat expansion diseases are a group of genetic disorders resulting from an increase in size or expansion of a specific array of tandem repeats. It has been suggested that DNA secondary structures are responsible for this expansion. If this is so, we would expect that all unstable repeats should form such structures. We show here that the unstable repeat that causes progressive myoclonus epilepsy type-1 (EPM1), like the repeats associated with other diseases in this category, forms a variety of secondary structures. However, EPM1 is unique in that tetraplexes are the only structures likely to form in long unpaired repeat tracts under physiological conditions. (+info)
Studies of the operator region of the Staphylococcus aureus beta-lactamase operon.
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The repressor proteins BlaI and MecI bind similarly to the bla operator implicated in the regulation of beta-lactamase synthesis in Staphylococcus aureus. BlaI binds to two separate dyads but neither copper-phenanthroline footprinting nor dimethyl sulphate (DMS) methylation protection assays produced any evidence of a change in the geometry of the DNA between the two dyads. It is concluded that BlaI molecules bound at the dyads probably do not cause bending or looping of the intervening DNA. DMS protection assays of BlaI binding to the bla operator in vitro and in vivo gave similar results so that it is tentatively concluded that the in vitro results are an accurate reflection of the in vivo situation. Deletion of the dyad nearest to the blaZ gene resulted in decreased synthesis of the chloramphenicol acetyltransferase reporter protein synthesized from the blaZ promoter/translation initiator. Explanations for this are considered. (+info)
RNA polymerase III transcription complexes on chromosomal 5S rRNA genes in vivo: TFIIIB occupancy and promoter opening.
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Quantitative analysis of multiple-hit potassium permanganate (KMnO(4)) footprinting has been carried out in vivo on Saccharomyces cerevisiae 5S rRNA genes. The results fix the number of open complexes at steady state in exponentially growing cells at between 8 and 17% of the 150 to 200 chromosomal copies. UV and dimethyl sulfate footprinting set the transcription factor TFIIIB occupancy at 23 to 47%. The comparison between the two values suggests that RNA polymerase III binding or promoter opening is the rate-limiting step in 5S rRNA transcription in vivo. Inhibition of RNA elongation in vivo by cordycepin confirms this result. An experimental system that is capable of providing information on the mechanistic steps involved in regulatory events in S. cerevisiae cells has been established. (+info)
Sequences upstream of the branch site are required to form helix II between U2 and U6 snRNA in a trans-splicing reaction.
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Three different base paired stems form between U2 and U6 snRNA over the course of the mRNA splicing reaction (helices I, II and III). One possible function of U2/U6 helix II is to facilitate subsequent U2/U6 helix I and III interactions, which participate directly in catalysis. Using an in vitro trans-splicing assay, we investigated the function of sequences located just upstream from the branch site (BS). We find that these upstream sequences are essential for stable binding of U2 to the branch region, and for U2/U6 helix II formation, but not for initial U2/BS pairing. We also show that non-functional upstream sequences cause U2 snRNA stem-loop IIa to be exposed to dimethylsulfate modification, perhaps reflecting a U2 snRNA conformational change and/or loss of SF3b proteins. Our data suggest that initial binding of U2 snRNP to the BS region must be stabilized by an interaction with upstream sequences before U2/U6 helix II can form or U2 stem-loop IIa can participate in spliceosome assembly. (+info)
Neuronal BC1 RNA structure: evolutionary conversion of a tRNA(Ala) domain into an extended stem-loop structure.
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By chemical and enzymatic probing, we have analyzed the secondary structure of rodent BC1 RNA, a small brain-specific non-messenger RNA. BC1 RNA is specifically transported into dendrites of neuronal cells, where it is proposed to play a role in regulation of translation near synapses. In this study we demonstrate that the 5' domain of BC1 RNA, derived from tRNA(Ala), does not fold into the predicted canonical tRNA cloverleaf structure. We present evidence that by changing bases within the tRNA(Ala) domain during the course of evolution, an extended stem-loop structure has been created in BC1 RNA. The new structural domain might function, in part, as a putative binding site for protein(s) involved in dendritic transport of BC1 RNA within neurons. Furthermore, BC1 RNA contains, in addition to the extended stem-loop structure, an internal poly(A)-rich region that is supposedly single stranded, followed by a second smaller stem-loop structure at the 3' end of the RNA. The three distinct structural domains reflect evolutionary legacies of BC1 RNA. (+info)
Hexosaminidase inhibitors as new drug candidates for the therapy of osteoarthritis.
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BACKGROUND: Articular cartilage from patients with osteoarthritis is characterized by a decreased concentration and reduced size of glycosaminoglycans. Degeneration of the cartilage matrix is a multifactorial process, which is due in part to accelerated glycosaminoglycan catabolism. Recently, we have demonstrated that hexosaminidase represents the dominant glycosaminoglycan-degrading glycosidase released by chondrocytes into the extracellular compartment and is the dominant glycosidase in synovial fluid from patients with osteoarthritis. Inhibition of hexosaminidase activity may represent a novel approach to the prevention of cartilage matrix glycosaminoglycan degradation and a potentially new strategy to treat osteoarthritis. RESULTS: We have synthesized and investigated a series of iminocyclitols designed as transition-state analog inhibitors of human hexosaminidase, and demonstrated that the five-membered iminocyclitol 4 expresses the strongest inhibitory activity with K(i)=24 nM. Inhibition of hexosaminidase activity in human cultured articular chondrocytes and human chondrosarcoma cells with iminocyclitol 4 resulted in accumulation of hyaluronic acid and sulfated glycosaminoglycans in the cell-associated fraction. Similarly, incubation of human cartilage tissue with iminocyclitol 4 resulted in an accumulation of glycosaminoglycans in the pericellular compartment. CONCLUSIONS: Inhibition of hexosaminidase activity represents a new strategy for preventing or even reversing cartilage degradation in patients with osteoarthritis. (+info)
A family of novel, acidic N-glycans in Bowes melanoma tissue plasminogen activator have L2/HNK-1-bearing antennae, many with sulfation of the fucosylated chitobiose core.
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A family of about 20 novel acidic bi- and tri-antennary N-glycans, amounting to almost half those expressed on Bowes melanoma tissue-plasminogen activator (t-PA) were found to possess Galbeta1-->4GlcNAcbeta1-->, sulfated and sialylated GalNAcbeta1-->4GlcNAcbeta1--> or sulfated GlcAbeta1--> 3Galbeta1-->4GlcNAcbeta1--> antennae, of which those containing sulfated GlcA, depicting the L2/HNK-1 carbohydrate epitope, were preferentially located on the 6 arm. A proportion of the glycans were highly charged, because of multiple and variously distributed sulfation, some of which was located on the fucosylated chitobiose core. Multiple expression of the L2/HNK-1 epitope on a single glycan was observed. The most abundant compound was a biantennary glycan carrying sulfated GlcA on the 6-branched antenna and an alpha2-->6 sialylated GalNAc on the other. The N-glycosylation sequon containing Asn448, which is known to express all of the sulfate-carrying N-glycans contains, unusually, an arginine residue. An electrostatic interaction between this cationic amino acid and the core-sulfate group of the N-glycan is proposed to reduce mobility of the carbohydrate in the region of the t-PA active site. Because of the 'brain-type' nature of the N-glycans described in this neuro-ectodermal cell line, the possibility of neural t-PA interacting with the L2/HNK-1-recognizing molecule, laminin, of the central nervous system extracellular matrix is discussed. (+info)