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. 2016 Nov 11;14(10):1353–1363. doi: 10.1080/15476286.2016.1256534

Figure 3.

Figure 3.

The small mRNA rreB is in the insoluble fraction and is strongly bound to the 30S ribosomal subunit in B. japonicum. (A) Northern blot analysis of total RNA (T) and RNA isolated from the P100 (P) and S100 (S) fraction using probes directed against the 5′-UTR (5′-probe), the sORF (ORF-probe) and the 3′-UTR (3′-probe) of rreB as indicated; the same membrane was re-hybridized. Main bands of approximately 165 nt and 60 nt were detected (marked at the left side). Their lengths were estimated by hybridization of the membrane with probes directed against the length standards 6S RNA (160 nt), 5S rRNA (120 nt) and tRNA-Arg (79 nt) (not shown). (B) Northern blot analysis of tRNA-Arg and 5S rRNA. The membrane shown in A) was re-hybridized. (C) Separation of the P100 fraction through a 5% to 40% sucrose density gradient. The absorption of the gradient fractions at 260 nm is shown. Fractions which were further analyzed are marked with arrows. (D) Analysis of RNA isolated from fractions 3, 10 and 14 of the sucrose density gradient by electrophoresis. RNA was separated in urea-containing 10% polyacrylamide gel and stained with ethidium bromide (shown is a negative image). Distinct bands corresponding to the large 23S rRNA fragment (23S rRNA), 16S rRNA, the 5.8S rRNA-like short 5′-fragment of 23S rRNA, 5S rRNA and tRNAs are indicated. Fragmentation of 23S rRNA in Bradyrhizobium japinicum was described previously.40,41 (E) Northern blot hybridization of RNA from the indicated sucrose density gradient fractions with probes directed against the sORF of rreB, tRNA-Arg and 5S rRNA.