Small RNAs and Protein Localization in Bacterial Development and Antimicrobial Drug Discovery
In order for bacteria to control cell cycle and developmental programs, they must be able to express the appropriate proteins at the specific time and in the specific place where they are needed. Temporal and spatial regulation of proteins are achieved through multiple, often redundant, mechanisms. We have two main areas of interest: temporal regulation of protein expression mediated by tmRNA, and mechanisms of protein localization in bacteria.
We are investigating a mechanism of translational control mediated by a very unusual small RNA, called tmRNA or SsrA RNA. tmRNA is a highly abundant RNA found in all bacteria. It alters the expression of substrate proteins by intervening during translation of the mRNA to target the nascent polypeptide for proteolysis, and to release the translating ribosomes from the mRNA. We have found that tmRNA is required for regulation of the cell cycle and development in Caulobacter crescentus, and we are using biochemical and genetic approaches to understand the molecular events which underlie the tmRNA mechanism and to understand the role of tmRNA in bacterial differentiation and physiology.
Because the tmRNA pathway is required for virulence in many pathogenic bacteria, it is an attractive target for antimicrobial drug discovery. In collaboration with the laboratory of Stephen Benkovic in the Chemistry Department, we have established methods for selecting inhibitors of the tmRNA pathway in vivo. Inhibitors of several components of the pathway have been isolated from a library of cyclic peptides produced using the SICLOPPS technology. These cyclic peptides have antibacterial activity and are lead compounds for antibiotic development.
In addition to temporal regulation, proteins are also controlled by altering their location within the cell. Several key regulatory and structural proteins have specific cellular addresses in bacteria, but it is not known how many proteins are localized in a bacterium, or how most of these proteins are targeted to the proper locations. We are using bacterial genetics and epifluorescence microscopy to identify localized proteins and to dissect the localization signals within these proteins.
Caulobacter crescentus- Differentiation and the Cell Cycle
Caulobacter is an ideal organism to study bacterial differentiation and cell-cycle regulation, since each round of the cell cycle includes an obligatory differentiation event (Fig. 1). Each cell division results in two cell types with different fates: a stalked cell which immediately begins another round of cell division, and a swarmer cell which cannot divide or initiate DNA replication until it differentiates into a new stalked cell.
A pure population of swarmer cells can easily be isolated, and this population will pass synchronously through the cell cycle. This synchronization procedure allows detailed study of the cell cycle, as well as the morphological differentiation during the swarmer-to-stalked cell transition. In addition, because each Caulobacter cell is spatially asymmetric, it is easy to tell one end of the cell from the other. This asymmetry greatly facilitates localization studies by allowing a more precise definition of the location of a protein.
Control of Translation by tmRNA
tmRNA has properties of both a tRNA and an mRNA. The 5’ and 3’ ends of the RNA fold into a structure which resembles alanyl tRNA, and SsrA can be charged with alanine by alanyl-tRNA synthetase. A separate part of the molecule contains a short open reading frame. tmRNA uses its unusual structure to add a peptide tag to the C-terminus of proteins as they are being translated, and this peptide tag targets the protein for rapid degradation in the cell. In this remarkable mechanism (Fig. 2), tmRNA acts first like a tRNA, entering the ribosomal A-site and becoming a substrate for transpeptidation. In this reaction, the alanine charged to the 3’ end of tmRNA is added to the C terminus of the nascent polypeptide. The reading frame then switches from the mRNA to the open reading frame within tmRNA, resulting in the addition of the tmRNA-encoded peptide tag to the C-terminus of the substrate protein. The peptide tag contains multiple proteolytic determinants which targets the substrate protein for rapid degradation by a number of intracellular proteases.
In Caulobacter, a deletion of tmRNA results in a delay in the initiation of DNA replication during the swarmer-to-stalked cell transition. By identifying the substrates of tmRNA and determining how these substrates are responsible for the cell-cycle phenotype, we aim to understand the role of tmRNA in cell-cycle regulation and bacterial differentiation. We have also found that the levels of tmRNA are regulated with respect to the cell cycle by transcription and RNA degradation. By studying the mechanism of regulation of tmRNA activity, we will identify new cell-cycle regulatory factors, and understand how tmRNA is integrated into the Caulobacter regulatory network.
Cyclic Peptide Inhibitors of tmRNA as Antibiotic Lead Compounds
Cyclic peptides can be produced in bacteria using a technology developed in the Benkovic lab called Split Intein Circular Ligation of Proteins and Peptides (SICLOPPS). From a library of randomized cyclic peptides, we have selected molecules that inhibit tagging by tmRNA or proteolysis of the tagged proteins. These cyclic peptides inhibit the reaction in vitro and kill Caulobacter crescentus cells when added to growing cultures. After optimizing the size and sequence of these peptides, they will be tested for antibacterial activity against pathogens of medical and military interest in preparation for future antibiotic development.
Protein Localization in Bacteria
We have used a genetic screen to identify localized proteins in Caulobacter crescentus. Early results from this screen have identified several known and previously hypothetical proteins with interesting localization patterns. Further characterization of these localized proteins will address the questions of why individual proteins are localized, and how localization affects function. This work will shed light on the fundamental questions of how many proteins are localized in a bacterial cell, how these proteins are targeted to the proper location, and how localization is coupled to protein function.