In the bustling microscopic factories of bacteria, the manufacturing of proteins is a meticulous process initiated by the unwinding of double-stranded DNA. This is the first step in a complex dance of transcription, where RNA is synthesized as a vital intermediary, eventually leading to the production of proteins that the bacterium requires at any given moment. Overseeing this intricate process are entities known as sigma factors, akin to line supervisors, each variety with its own architectural and genetic signature, broadly categorized into four known groups.

Yet, within this microbial world, one sigma factor has long stood out as an enigma. SigI, as it is designated, plays a role in a range of regulatory responses but defies the conventional group classifications. Researchers at the Qingdao Institute of Bioenergy and Bioprocess Technology, part of the Chinese Academy of Sciences, have unveiled that SigI's distinctiveness is not merely superficial; it is profound enough to warrant the creation of a new classification group entirely.

Their study, detailed recently in Nature Communications, sheds light on a previously obscure corner of genetic regulation, with implications that could ripple through the field of gene regulation tool design.

Sigma factors, in their role, are tasked with identifying specific promoters—regions at the very beginning of a DNA sequence that signals the need for a particular transcript. The researchers were intrigued by the observation that different SigI factors within the same bacterium can bind to various promoters, ensuring precise recognition.

For over two decades, since SigI was first identified, the molecular underpinnings of how it recognizes specific promoters during transcription remained a mystery. "The existence of multiple SigIs and their role regulating the expression of a super multienzyme complex, called cellulosome, in some bacteria for efficient degradation of plant biomass raised further questions about the mechanism of specificity between different SigIs," explained Prof. Feng Yingang from QIBEBT, co-corresponding author of the study.

To unravel this mystery, the team reconstructed two transcription open complexes—one with SigI1 and another with SigI6—using RNA polymerase from the bacterium C. thermocellum combined with SigI factors from E. coli and synthesized promoters. They employed cryo-electron microscopy to discern the structures of these complexes and conducted experimental analyses to verify their functions.

Through their structural and functional analyses, the researchers not only categorized SigI but also decoded the specificity of its functions. They discovered that SigI factors from cellulosome-producing bacteria possess a novel, previously unidentified mode of recognizing bacterial transcriptional promoters, marking them as a new, distinct class of sigma factors.

The revealed structures indicated that SigI possesses two structural features, one of which is absent in other known sigma factors, facilitating a unique recognition mode for a specific promoter with a -35 element. "SigI promoter recognition of the -35 element differs completely from that of other sigma factors in this family," stated Feng. Moreover, the structures suggested that SigIs could identify another promoter region, the -10 element, using different features from those utilized by other sigma factors.

The research team is poised to delve deeper into the workings of SigI factors in regulating cellulosomes in C. thermocellum.

"Although we have gained some knowledge of the SigI system in transcriptional regulation of cellulosome, the understanding of cellulosome regulation is still far from complete," Feng acknowledged. With about 80 cellulosomal components in C. thermocellum, only a fraction has been confirmed to be under the SigI system's influence. The ultimate aim is to demystify the molecular mechanisms that control cellulosomes. With this knowledge, the team hopes to engineer cellulosome-producing bacteria to create more efficient biofactories for applications spanning bioenergy, synthetic biology, and biotechnology.