Mechanisms of Processive Antitermination in Gram-Positive Bacteria

Since Theodor Escherich first described "bacteria coli commune" in 1885, which was later renamed Escherichia coli, it has become the most studied organism at the molecular level. The intensity of this focus has many benefits, but also poses risks, as assumptions from using a single bacterium as a gold standard can be misleading. While much is now known about gene expression in E. coli, the rules differ significantly for Bacillus subtilis and other Gram-positive bacteria, particularly in transcription and translation mechanisms. For example, a model for E. coli gene expression has emerged that proposes that the transcription elongation complex (TEC) is physically linked to a leading ribosome and that the two molecular machines proceed at a similar overall rate in a process referred to as transcription-translation coupling. During active translation, the coupled machines block access to the Rho termination factor; however, when a stop codon ends transcription-translation coupling, temporarily divorcing their interactions, the Rho termination factor gains access to an elongation factor (NusG) that is associated with the TEC and triggers transcription termination. This is now the textbook depiction of gene expression in E. coli, and it has been assumed that it applies to most genes and operons. However, for some noncoding operons, such as those that are responsible for synthesizing ribosomal RNAs or CRISPR RNAs, the E. coli TEC is modified into what are referred to as antitermination complexes. These antitermination complexes are formed through interactions with transcription elongation factors that modify the TEC such that it becomes resistant to the Rho termination factor, thereby allowing uninterrupted transcription of the ribosomal and CRISPR RNAs. But recent data suggest this is not what happens in B. subtilis. The B. subtilis TEC proceeds at a rate that is twice as fast as the ribosome, and it is now believed that transcription and translation remain uncoupled in B. subtilis and most other Gram-positive bacteria. Since most structural and biochemical data on the regulation of transcription elongation are based on data from E. coli, there is a striking gap in knowledge regarding how the TEC is regulated in B. subtilis and other Gram-positive bacteria. We recently discovered two novel antitermination mechanisms that are widespread in Gram-positive bacteria and that regulate transcription elongation through unknown mechanisms. One of these antitermination mechanisms regulates operons encoding biofilm expopolysaccharides and the other controls the expression of operons that synthesize antibiotics and other secondary metabolites. In this project, we will elucidate the molecular mechanisms underlying these antitermination strategies. Our data from this investigation will be broadly relevant to the regulation of biofilm formation and antibiotic production by Gram-positive bacteria. Moreover, it will provide a much more accurate textbook understanding of the mechanisms of transcription elongation in bacteria. Project Number: 1R35GM161408-01 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of General Medical Sciences (NIGMS) | Principal Investigator: Wade Winkler | Institution: UNIV OF MARYLAND, COLLEGE PARK, COLLEGE PARK, MD | Award Amount: $381,240 | Activity Code: R35 | Study Section: Maximizing Investigators' Research Award - F Study Section[MRAF] View on NIH RePORTER: https://reporter.nih.gov/project-details/11259954