Top2 cannot act via Sgo1 recruitment in order to fulfill its function in Ipl1 recruitment

ith iodo-acetic acid and then analysed by electrophoresis. Panel D, the carboxylation reaction was carried out for 10 mnin at the temperatures indicated above the gel lanes. 4. 1′ thi2 R op iL hit affinity, RNAI and RNAII lead us to propose a model where each subunit of the dimer recognizes similar ribonucleotide sequences in stem structures and functions as an adaptor that correctly positions the two RNAs for loop interaction. To gain further information about the geometry PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19828691 of this interaction and to identify the amino acid side chains that are responsible for RNA recognition, we have systematically modified the amino acids whose side chains are exposed to the solvent and we have determined whether these altered proteins still maintain their function. For mutant isolation and overexpression we have made use of an expression plasmid containing the replication origin of phage fl This allows us to prepare. the plasmid chromosome in a single stranded form for oligonucleotide directed mutagenesis and for mutant sequencing. pEX43 has the Rop gene under the control of the PL promoter of phage X and in the absence of an active X cI repressor directs the synthesis of large amounts of Rop protein. A further useful feature of this vector consists in the presence, between the PL promoter and the Rop structural gene, of a strong promoter for the RNA polymerase of phage T7. We have exploited this feature to transcribe efficiently the Rop gene in vitro and to produce small amounts of specifically labelled Rop variants in an Escherichia coli in vitro translation system. Rop function was assayed in two ways. As a first quick test we exploited the ability 623 L.Castagnoli et al. of Rop to decrease the expression of genes transcriptionally fused to the first 110 nt of the primer precursor. L76, an E. coli strain containing a fusion between the 3-galactosidase structural gene and the initial nucleotides of the primer transcript forms red colonies on MacConkey plates. In the presence of a ColEl type plasmid encoding an active Rop gene, however, fl-galactosidase levels are decreased and the strain forms white colonies. We used this test as a screening procedure that allowed us to identify colonies harbouring defective proteins by direct inspection of colony colour on the plate. The second test involved estimating plasmid copy number by determining the ampicillin concentration at which the colony forming ability of a strain is decreased by a factor of 10. Defective Rop proteins could in principle derive either from correctly folded peptides in which an essential amino acid side chain has been altered, or from mutant proteins that do not assume the correct three-dimensional conformation. From our experience, most of the mutants that belong to this latter class are degraded by E. coli proteases. Thus, to characterize further the functionally negative mutants that we have isolated, we tested whether we could recover Rop protein from a cell extract containing an overproducer plasmid directing the synthesis of the defective protein. Mutant Rop proteins that could be overproduced were assumed to be correctly folded and provisionally classified as ‘functional’ mutants, while mutants that could not be recovered from E.coli extracts were classified as ‘folding’ mutants. In all the cases in which this assumption was tested by biochemical, spectroscopic or Scopoletin crystallographic techniques it proved to be correct. As a second, more direct test for Rop folding we devised a met