(A) Template DNA sequence where the X region contains the pG4 or buG4 sequences. The T7 promoter region where the T7 RNA polymerase will bind and the TSS where the transcription will start, are highlighted in the template sequence. The bulge positions 1, 2, 3, 4, 5, 6, 7, and 8 are represented as dots. When T7 RNA polymerase stops in front of the G-rich region a 15 nt short RNA will be produced, whereas, when it runs off till the end of the sequence a 55 nt RNA will be produced. Template sequences are shown in Supplementary Table S2. (B) Transcript production of the linear, pG4 and buG4 containing template sequences in the presence of 100 mM KCl and 10 wt% PEG200 as crowding condition. Denaturing gel electrophoresis was used to identify the products of transcription reactions carried out for 120 min at 37°C. The marked lanes represent the transcript formation from the template DNA containing Linear, pG4, bT₁-1, bT₁-2, bT₁-3, bT₁-4, bT₁-5, bT₁-6, bT₁-7, and bT₁-8. M is the size marker. R1 and R2 are the 15 nt and 20 nt RNA sequences, respectively as markers. The 15 nt arrested bands from pG4 and buG4 containing templates are marked with stars. (C) Transcription arrested efficiency (TE_arrest) for the template DNA containing pG4 and single bulge buG4s, calculated as the ratio of arrested transcript-1 intensity to total transcript intensity. TE_arrest values below 10% are considered as background, hence considered as no arrest.