A large class of bacterial RNA polymerase (RNAP) from low-G+C-content Gram-positive bacterial strains, such as the major human pathogen Staphylococcus aureus, not only contain five conserved subunits (a(I), a(II), beta, beta' and u), but also has a delta subunit. Despite being first identified as unique, Grampositive specific component of RNAP apoenzyme more than 30 years ago and reported to be essential for transcription, the structural basis and molecular mechanism of delta subunit in the regulation of transcription remain poorly understood. Here, we performed structural analyses, site-directed mutagenesis and biochemical assays to uncover the interactions of S. aureus d subunit with RNAP core enzyme and DNA towards the understanding of its role in transcription regulation. Microscale thermophoresis (MST) and electrophoretic mobility shift assay (EMSA) of the wild-type and mutated S. aureus d subunit revealed the N-terminal domain of d subunit directly binds to the beta' jaw of S. aureus RNAP (SauRNAP), identified the key amino acid residues (F58, D61, D65, R67 and W81) of d subunit involving in the binding with SauRNAP core enzyme, and uncovered the d subunit C-terminal domain interferes with the interaction between DNA and SauRNAP core enzyme, by which transcription is regulated. Our results provide an excellent starting point for understanding the unique regulatory role and physiological function of d subunit on transcription regulation in Gram-positive bacteria. (C) 2021 Elsevier Inc. All rights reserved.