FACT and Ubp10 collaborate to modulate H2B deubiquitination and nucleosome dynamics.

Monoubiquitination of histone H2B (H2B-Ub) plays a role in transcription and DNA replication, and is required for normal localization of the histone chaperone, FACT. In yeast, H2B-Ub is deubiquitinated by Ubp8, a subunit of SAGA, and Ubp10. Although they target the same substrate, loss of Ubp8 and Ubp10 cause different phenotypes and alter the transcription of different genes. We show that Ubp10 has poor activity on yeast nucleosomes, but that the addition of FACT stimulates Ubp10 activity on nucleosomes and not on other substrates. Consistent with a role for FACT in deubiquitinating H2B in vivo, a FACT mutant strain shows elevated levels of H2B-Ub. Combination of FACT mutants with deletion of Ubp10, but not Ubp8, confers increased sensitivity to hydroxyurea and activates a cryptic transcription reporter, suggesting that FACT and Ubp10 may coordinate nucleosome assembly during DNA replication and transcription. Our findings reveal unexpected interplay between H2B deubiquitination and nucleosome dynamics.

Malonate degradation in Acinetobacter baylyi ADP1: operon organization and regulation by MdcR.

Transcriptional regulators in the LysR or GntR families are typically encoded in the genomic neighbourhood of bacterial genes for malonate degradation. While these arrangements have been evaluated using bioinformatics methods, experimental studies demonstrating co-transcription of predicted operons were lacking. Here, transcriptional regulation was characterized for a cluster of mdc genes that enable a soil bacterium, Acinetobacter baylyi ADP1, to use malonate as a carbon source. Despite previous assumptions that the mdc-gene set forms one operon, our studies revealed distinct promoters in two different regions of a nine-gene cluster. Furthermore, a single promoter is insufficient to account for transcription of mdcR, a regulatory gene that is convergent to other mdc genes. MdcR, a LysR-type transcriptional regulator, was shown to bind specifically to a site where it can activate mdc-gene transcription. Although mdcR deletion prevented growth on malonate, a 1 nt substitution in the promoter of mdcA enabled MdcR-independent growth on this carbon source. Regulation was characterized by methods including transcriptional fusions, quantitative reverse transcription PCR, reverse transcription PCR, 5'-rapid amplification of cDNA ends and gel shift assays. Moreover, a new technique was developed for transcriptional characterization of low-copy mRNA by increasing the DNA copy number of specific chromosomal regions. MdcR was shown to respond to malonate, in the absence of its catabolism. These studies contribute to ongoing characterization of the structure and function of a set of 44 LysR-type transcriptional regulators in A. baylyi ADP1.

Close and Allosteric Opening of the Polypeptide-Binding Site in a Human Hsp70 Chaperone BiP.

Binding immunoglobulin protein (BiP), an essential and ubiquitous Hsp70 chaperone in the ER, plays a key role in protein folding and quality control. BiP contains two functional domains: a nucleotide-binding domain (NBD) and a substrate-binding domain (SBD). NBD binds and hydrolyzes ATP; the substrates for SBD are extended polypeptides. ATP binding allosterically accelerates polypeptide binding and release. Although crucial to the chaperone activity, the molecular mechanisms of polypeptide binding and allosteric coupling of BiP are poorly understood. Here, we present crystal structures of an intact human BiP in the ATP-bound state, the first intact eukaryotic Hsp70 structure, and isolated BiP-SBD with a peptide substrate bound representing the ADP-bound state. These structures and our biochemical analysis demonstrate that BiP has a unique NBD-SBD interface that is highly conserved only in eukaryotic Hsp70s found in the cytosol and ER to fortify its ATP-bound state and promote the opening of its polypeptide-binding pocket.