Transcription and translation are the core processes of gene expression. In bacteria, these two processes occur in the same cellular compartment and at the same time, and in many cases, are functionally coordinated and physically coupled. This phenomenon is known as transcription-translation coupling.

In molecular assemblies of tight transcription-translation complexes (TTC-B), transcription elongation factor NusG or its paralog RfaH forms a bridge between RNA polymerase (RNAP) and ribosome, and transcription elongation factor NusA optionally forms a second bridge between RNAP and ribosome. However, TTC-B accommodates only short mRNA spacer. When the ribosome has not yet caught up with RNAP, a long-range coupling mode also exists, but its structural mechanism remains elusive.

In a study published in PNAS, a research team led by WANG Chengyuan from the Shanghai Institute of Materia Medica (SIMM) of the Chinese Academy of Sciences, and Richard H. Ebright from Rutgers University, defined a new structural state named long-range coupled complex (TTC-LC) for transcription-translation coupling.

Using cryo-electron microscopy reconstruction, the researchers resolved high-resolution structures of NusG/RfaH-mediated transcription-translation complexes with long mRNA spacers. They found that when mRNA spacer exceeded 12 codons, RNAP underwent a ~60° rotation and a ~70 Å translation relative to the ribosome. This rearrangement created a ~70 Å gap between the two molecular machines, within which the mRNA folded into a loop structure, accommodating the extended spacer sequence.

Importantly, TTC-LC could shift into TTC-B after ribosomes caught up with RNAP. TTC-B also transformed back into TTC-LC when RNAP moved ahead of ribosomes. These observations showed that TTC-LC is a functional intermediate in assembling and disassembling TTC-B, mediating pre-TTC-B transcription-translation coupling before a ribosome catches up to RNAP, and mediating post-TTC-B transcription-translation coupling after a ribosome stops moving and RNAP continues moving.

Furthermore, in vitro biochemical assays revealed distinct regulatory effects on transcription termination. TTC-B strongly blocked hairpin-dependent termination. TTC-LC produced only mild inhibition on this termination. Notably, both complexes efficiently suppressed Rho-dependent termination. These findings shed new light on the regulatory roles of transcription-translation coupling in gene expression.

This study describes the molecular basis of long-range transcription-translation coupling in bacteria for the first time. The dynamic switch between TTC-LC and TTC-B explains real-time gene expression regulation in bacteria. The structural data provide new design ideas for synthetic transcription-translation systems, and potential molecular targets for developing new antibacterial drugs.