Extended Data Fig. 5: Details of ORF extension constructs and transcription terminator readthrough vs. stop-to-stem distances.
From: Functionally uncoupled transcription–translation in Bacillus subtilis
a, Sequence for terminators T1 and T2 for three variants (T1+: pupG original terminator, T1-: disrupted pupG terminator, ORF extension: original pupG with upstream ORF extended inside the loop of the terminator). For T1 and T2, blue and grey shading, respectively, marks the position of the terminator hairpin stems, with free energy of folding ∆G indicated. Black stars indicate introduced mutations. Downward carets (▼) indicate the position of the 3′ ends associated with intrinsic terminators as determined by Rend-seq. Red dashed line indicates the complementary region of the northern blot probe to the readthrough product. b, Terminator readthrough fraction (defined as the Rend-seq read density after terminator divided by read density upstream of terminator, see ref.35 for details) as a function of stop-to-stem distance for E. coli intrinsic terminators from Fig. 2 for which readthrough could be reliably estimated (n = 392). Terminators with stop-to-stem distance d ≤ 12 nt are highlighted in red. c, Cumulative distribution function of terminator readthrough for terminators far (black, d>12 nt) from and close (red, d ≤ 12 nt) to stop codons. Terminator close to genes have significantly more readthrough (less termination), P < 10−3 (q30d>12 and q30d ≤ 12 indicate the 30th percentile in the readthrough distribution for the two categories of terminators, with fold-change F30: = q30d ≤ 12/ q30d>12, P-value determined as the fraction of bootstrap random sub-samplings of the readthrough distributions with q30d>12 > q30d ≤ 12, Methods) d, Terminator readthrough as a function of ∆GU, the U-tract DNA/RNA hybrid free energy (measure of U-tract quality, with larger ∆GU corresponding to U-rich U-tract). Grey shading indicates cutoff (∆GU>-5 kcal/mol) to select good U-tract terminators. e, Same as c, but restricting to good U-tract terminators, still showing significantly less termination for terminators near ORF, P < 10−3 (same as above, Methods). f-i, same as b–e, but with terminators from B. subtilis. Terminators close to ORF do not show less readthrough than their gene-distal counterparts (P > 0.3, p-value determined with same strategy as above, Methods).
