Overlapping recognition determinants within the ssrA degradation tag allow modulation of proteolysis.

The ssrA tag, an 11-aa peptide added to the C terminus of proteins stalled during translation, targets proteins for degradation by ClpXP and ClpAP. Mutational analysis of the ssrA tag reveals independent, but overlapping determinants for its interactions with ClpX, ClpA, and SspB, a specificity-enhancing factor for ClpX. ClpX interacts with residues 9-11 at the C terminus of the tag, whereas ClpA recognizes positions 8-10 in addition to residues 1-2 at the N terminus. SspB interacts with residues 1-4 and 7, N-terminal to the ClpX-binding determinants, but overlapping the ClpA determinants. As a result, SspB and ClpX work together to recognize ssrA-tagged substrates efficiently, whereas SspB inhibits recognition of these substrates by ClpA. Thus, dissection of the recognition signals within the ssrA tag provides insight into how multiple proteins function in concert to modulate proteolysis.

Three Escherichia coli heat shock proteins are required for P1 plasmid DNA replication: formation of an active complex between E. coli DnaJ protein and the P1 initiator protein.

DNA containing the plasmid origin of bacteriophage P1 is replicated in vitro by a protein fraction prepared from uninfected Escherichia coli supplemented with purified P1 RepA protein. It has previously been shown that the reaction required the E. coli DnaA initiator protein, the DnaB helicase, DnaC protein, RNA polymerase, and DNA gyrase. I show here that three E. coli heat shock proteins, DnaJ, DnaK, and GrpE, are directly involved in P1 plasmid replication. Purified DnaJ, DnaK, and GrpE proteins were required to stimulate P1 plasmid ori DNA-dependent replication in in vitro complementation assays in which the host protein fractions were prepared from cells mutated in the corresponding gene. I have also found that the DnaJ and RepA proteins form a complex. This complex exists in crude cell extracts and can be isolated as a molecular species of about 160,000 Da containing one dimer of DnaJ protein and one dimer of RepA. The complex can also be reconstituted by mixing purified DnaJ and RepA proteins. These results imply that the DnaJ-RepA complex, DnaK, and GrpE are directly involved in P1 plasmid replication.

Mini-P1 plasmid replication: the autoregulation-sequestration paradox.

It has been proposed that the initiator protein RepA is rate limiting for mini-P1 plasmid replication, and that the role of the plasmid copy number control locus is to sequester the initiator and thus reduce replication. This proposal appears inconsistent with the observation that RepA is autoregulated, since the protein lost by sequestration should be replenished. A resolution of this autoregulation-sequestration paradox is possible if the sequestered RepA, unavailable for replication, is still available for promoter repression. We demonstrate that RepA binds to the control locus and to the promoter region simultaneously, causing the intervening DNA to loop. DNA looping could provide the requisite mechanism by which RepA bound to the control locus might exert repression.

Replication of mini-P1 plasmid DNA in vitro requires two initiation proteins, encoded by the repA gene of phage P1 and the dnaA gene of Escherichia coli.

We have developed an in vitro DNA-replication system that replicates exogenously added mini-P1 plasmid DNA. The system consists of purified P1 RepA protein and a partially purified mixture of Escherichia coli replication proteins. It is essentially the same as that described for the replication of oriC plasmid DNA [Fuller, R.S., Kaguni, J.M. & Kornberg, A. (1981) Proc. Natl. Acad. Sci. USA 78, 7370-7374]. Mini-P1 DNA replication requires the E. coli DnaA initiation protein in addition to the P1 RepA initiation protein. The reaction is inhibited by rifampicin, novobiocin, and antibody to DnaB, suggesting the involvement of RNA polymerase, DNA gyrase, and DnaB protein. Replication is initiated in the region of the P1 origin of replication and proceeds unidirectionally as determined by electron microscopy. Thus, the in vitro system mimics the essential features of mini-P1 replication as suggested by genetic studies.

Characterization of the DNA binding domain of bacteriophage lambda O protein.

The lambda O and P gene products are required for the initiation of lambda DNA replication. In order to study the biochemistry of this process, we have constructed plasmids that carry the lambda O gene, P gene, and half of the O gene coding for the amino-terminal half of the O protein. Each is under the control of the inducible lambda promoter, PL. We have purified these three proteins from induced cells carrying the plasmids. Our results show that the amino-terminal portion of the O protein binds to the lambda origin of replication in a manner similar to the intact lambda O protein, demonstrating that the amino-terminal portion of O protein contains the DNA binding domain. Using chromatographic procedures, we have isolated a complex of lambda O and P proteins with lambda dv DNA. The amino-terminal portion of the O protein does not complex with P protein under the same conditions. This suggests that the specificity of the lambda O protein for P protein resides in the carboxyl-terminal half of the lambda O protein. Our results also show that, while the intact O protein is active in in vitro replication of lambda dv plasmid DNA, the amino-terminal portion of the O protein is inactive and is a competitive inhibitor of the lambda O protein in this reaction. These results confirm previous genetic observations that were interpreted as indicating a bifunctional structure for the lambda O protein with the amino-terminal domain recognizing the lambda origin of replication and the carboxyl-terminal domain interacting with the lambda P protein.