Recent discovery of the RNA/DNA hybrid G-quadruplexes (HQs) and their potential wide-spread occurrence in human genome during transcription have suggested a new and generic transcriptional control mechanism. The G-rich sequence in which HQ may form can coincide with that for DNA G-quadruplexes (GQs), which are well known to modulate transcriptions. Understanding the molecular interaction between HQ and GQ is, therefore, of pivotal importance to dissect the new mechanism for transcriptional regulation. Using a T7 transcription model, herein we found that GQ and HQ form in a natural sequence, (GGGGA)4, downstream of many transcription start sites. Using a newly-developed single-molecular stalled-transcription assay, we revealed that RNA transcripts helped to populate quadruplexes at the expense of duplexes. Among quadruplexes, HQ predominates GQ in population and mechanical stabilities, suggesting HQ may serve as a better mechanical block during transcription. The fact that HQ and GQ folded within tens of milliseconds in the presence of RNA transcripts provided justification for the co-transcriptional folding of these species. The catalytic role of RNA transcripts in the GQ formation was strongly suggested as the GQ folded >7 times slower without transcription. These results shed light on the possible synergistic effect of GQs and HQs on transcriptional controls.