Interplay of signal recognition particle and trigger factor at L23 near the nascent chain exit site on the Escherichia coli ribosome.

As newly synthesized polypeptides emerge from the ribosome, they interact with chaperones and targeting factors that assist in folding and targeting to the proper location in the cell. In Escherichia coli, the chaperone trigger factor (TF) binds to nascent polypeptides early in biosynthesis facilitated by its affinity for the ribosomal proteins L23 and L29 that are situated around the nascent chain exit site on the ribosome. The targeting factor signal recognition particle (SRP) interacts specifically with the signal anchor (SA) sequence in nascent inner membrane proteins (IMPs). Here, we have used photocross-linking to map interactions of the SA sequence in a short, in vitro-synthesized, nascent IMP. Both TF and SRP were found to interact with the SA with partially overlapping binding specificity. In addition, extensive contacts with L23 and L29 were detected. Both purified TF and SRP could be cross-linked to L23 on nontranslating ribosomes with a competitive advantage for SRP. The results suggest a role for L23 in the targeting of IMPs as an attachment site for TF and SRP that is close to the emerging nascent chain.

Sequence-specific interactions of nascent Escherichia coli polypeptides with trigger factor and signal recognition particle.

As nascent polypeptides exit the ribosomal tunnel they immediately associate with chaperones, folding catalysts, and targeting factors. These interactions are decisive for the future conformation and destination of the protein that is being synthesized. Using Escherichia coli as a model organism, we have systematically analyzed how the earliest contacts of nascent polypeptides with cytosolic factors depend on the nature and future destination of the emerging sequence using a photo cross-linking approach. Together, the data suggest that the chaperone trigger factor is adjacent to emerging sequences by default, consistent with both its placement near the nascent chain exit site and its cellular abundance. The signal recognition particle (SRP) effectively competes the contact with TF when a signal anchor (SA) sequence of a nascent inner membrane protein appears outside the ribosome. The SRP remains in contact with the SA and downstream sequences during further synthesis of approximately 30 amino acids. The contact with trigger factor is then restored unless another transmembrane segment reinitiates SRP binding. Importantly and in contrast to published data, the SRP appears perfectly capable of distinguishing SA sequences from signal sequences in secretory proteins at this early stage in biogenesis.

The Sec-independent function of Escherichia coli YidC is evolutionary-conserved and essential.

YidC plays a role in the integration and assembly of many (if not all) Escherichia coli inner membrane proteins. Strikingly, YidC operates in two distinct pathways: one associated with the Sec translocon that also mediates protein translocation across the inner membrane and one independent from the Sec translocon. YidC is homologous to Alb3 and Oxa1 that function in the integration of proteins into the thylakoid membrane of chloroplasts and inner membrane of mitochondria, respectively. Here, we have expressed the conserved region of yeast Oxa1 in a conditional E. coli yidC mutant. We find that Oxa1 restores growth upon depletion of YidC. Data obtained from in vivo protease protection assays and in vitro cross-linking and folding assays suggest that Oxa1 complements the insertion of Sec-independent proteins but is unable to take over the Sec-associated function of YidC. Together, our data indicate that the Sec-independent function of YidC is conserved and essential for cell growth.

SecB is a bona fide generalized chaperone in Escherichia coli.

It is known that the DnaK and Trigger Factor (TF) chaperones cooperate in the folding of newly synthesized cytosolic proteins in Escherichia coli. We recently showed that despite a very narrow temperature range of growth and high levels of aggregated cytosolic proteins, E. coli can tolerate deletion of both chaperones, suggesting that other chaperones might be involved in this process. Here, we show that the secretion-dedicated chaperone SecB efficiently suppresses both the temperature sensitivity and the aggregation-prone phenotypes of a strain lacking both TF and DnaK. SecB suppression is independent of a productive interaction with the SecA subunit of the translocon. Furthermore, in vitro cross-linking experiments demonstrate that SecB can interact both co- and posttranslationally with short nascent chains of both secretory and cytosolic proteins. Finally, we show that such cotranslational substrate recognition by SecB is greatly suppressed in the presence of ribosome-bound TF, but not by DnaK. Taken together, our data demonstrate that SecB acts as a bona fide generalized chaperone.

Expression of the mau gene cluster of Paracoccus denitrificans is controlled by MauR and a second transcription regulator.

The mau gene cluster of Paracoccus denitrificans constitutes 11 genes (10 are located in the transcriptional order mauFBEDACJGMN; the 11th, mauR, is located upstream and divergently transcribed from these genes) that encode a functional methylamine-oxidizing electron transport branch. The mauR gene encodes a LysR-type transcriptional activator essential for induction of the mau operon. In this study, the characteristics of that process were established. By using lacZ transcriptional fusions integrated into the genome of P. denitrificans, it was found that the expression of the mauR gene during growth on methylamine and/or succinate was not autoregulated, but proceeded at a low and constant level. The mauF promoter activity was shown to be controlled by MauR and a second transcriptional regulator. This activity was very high during growth on methylamine, low on succinate plus methylamine, and absent on succinate alone. MauR was overexpressed in Escherichia coli by using a T7 RNA polymerase expression system. Gel shift assays indicated that MauR binds to a 403 bp DNA fragment spanning the mauR-mauF promoter region. It is concluded from these results that the expression of the structural mau genes is dependent on MauR and its inducer, methylamine, as well as on another transcription factor. Both activators are required for high-level transcription from the mauF promoter. It is hypothesized that the two activators act synergistically to activate transcription: the effects of the two activators are not additive and either one alone activates the mauF promoter rather weakly.