Typhoon 5

For quantification of in vitro reconstituted ubiquitylation assays, the intensities of radiolabeled peptide or protein substrates and their ubiquitylated forms were imaged by scanning SDS-PAGE gels on a Typhoon 5 (Cytiva) and quantified using ImageQuant software. Experiments measuring the Michaelis constant K_m were fit using nonlinear regression on GraphPad Prism v10, while k_obs was analyzed using Mathematica v13.1. All data were measured in triplicate technical replicates. Binding assays were fit using one-phase association in GraphPad Prism v10 and performed in triplicate technical replicates, with data being represented as the average of each replicate, and the error representing the standard error. Statistical analysis and significance in the cellular PROTAC-dependent neo-substrate degradation assay was determined using an unpaired t-test with Welch’s correction. Statistical parameters that were reported in the figures are described in the corresponding figure legend.

A recombination-deficient Δ7 prrn strain was generated by moving Δ(recA-srl)306 srlR::Tn10 into SQZ10 (61 ) via P1 transduction, resulting in strain BRW246. Mutations to the leader and trailer region of rrsB were introduced into plasmid p278MS2 (62 (link)) to generate variants listed in Table 1. These plasmids were transformed into SQZ10 (or BRW246), and transformants were plated on sucrose (5%) to select against the resident plasmid pHKrrnC-sacB (54 (link)). Strains were verified by plasmid purification and sequencing. For growth rate measurements, overnight cultures were diluted 200-fold into fresh LB Amp, and growth was monitored at 37°C by measuring OD₆₀₀ as a function of time.
Variants of strain BRW246 (WT, ΔboxA, Δ1575–1598, ΔhA and ΔhB) were grown to mid-log phase and subjected to sucrose gradient sedimentation analysis as described (63 (link)). Fractions (0.5 ml) were collected across the gradient, and RNA from the pre-30S, 30S and 70S regions of the gradient was extracted and analyzed by denaturing PAGE as previously described (14 (link),64 (link)). Gels were stained with SYBR-Gold Nucleic acid stain (Invitrogen) and scanned using a Typhoon 5 (Cytiva). Data were quantified using ImageQuant (Cytiva).

RNA samples (5 μg) were separated by electrophoresis through 10% (for nuclease knockdown and overexpression experiments) or 15% (for dual reporter experiments) TBE-urea gels (Invitrogen). Following electrophoresis, the RNA was transferred to a nylon membrane (PerkinElmer). The membrane was dried overnight and UV-crosslinked. Pre-hybridization was carried out in Rapid-hyb Buffer (GE Healthcare) at 42°C. Probes were generated by end-labeling oligonucleotides (IDT) with γ-³²P ATP (PerkinElmer) using T4 PNK (NEB), and then probes were purified using Illustra Microspin G-50 columns (GE Healthcare) to remove unincorporated nucleotides. Upon purification, probes were boiled, cooled on ice and then added to the Rapid-hyb Buffer for hybridization. After hybridization, the membrane was washed in saline-sodium citrate (SSC) buffer. For probe sequences, see Supplementary Table S1. Washing conditions are as follows. U1 and U6: hybridization at 65°C, washes (twice in 2× SSC, twice in 0.33× SSC) at 60°C. 7SK and dual reporter probe: hybridization at 42°C, two washes in 5× SSC at 25°C and two washes in 1× SSC at 42°C. For the dual reporter probe, two additional washes in 0.1× SSC at 45°C were performed. The membrane was then exposed to a storage phosphor screen (GE Healthcare) and imaged on an Amersham Typhoon 5.