During RNA synthesis, RNA polymerase (RNAP) can temporally halt nucleotide addition by pausing. Transcriptional pausing plays a central role in regulation of transcription and may involve conformational changes in RNAP due to interactions of RNAP with intrinsic signals encoded in DNA and RNA, changes in translocation register, or both. However, basic mechanisms of transcriptional pausing and the role of RNAP translocation in regulation of transcript elongation are poorly understood. Here, we present work to investigate pause mechanisms, specifically how the pause signal is integrated/transmitted from the RNA exit channel of the enzyme to the enzymefs active site, and the contributions of RNAP translocation to pausing. We determined that the identity of RNA:DNA nucleotides in the active site are strong determinants of translocation bias, with the 3L RNA nucleotide favoring the pretranslocated state in the order U>C>A>G. Transcript elongation in bacteria is regulated in part by structures that form in the nascent RNA transcript and interact with RNAP in the RNA exit channel of the enzyme. A pause RNA hairpin structure makes direct contact with the flap domain of the RNAP fÀ subunit, and slows nucleotide addition by factors of 10-20. We investigated the effects of different length and type of duplexes in the RNAP exit channel on pausing and response to regulators. We show that 8-bp RNA duplexes can stimulate pausing and those with less than 8-bp duplexes do not give full hairpin effect. We also investigated how regulatory communication between the formation of secondary structure in the RNA exit channel and the active site of RNAP modulate the catalytic center function. The opening of the RNAP clamp has been proposed to occur during pausing and stabilize the pause state. Our findings indicate that the formation of the paused 8-bp RNA:RNA duplex in the RNA exit channel causes movement of RNAPfs clamp and the flap domains, which in turn inhibits RNAP translocation and the catalysis of rapid nucleotide transfer. Transcription factors like NusA and RfaH tune and provide additional function to this communication network by affecting the clamp movements, translocation, or both functions.