We are sorry, but NCBI web applications do not support your browser and may not function properly. More information

Results: 4

1.
FIGURE 3.

FIGURE 3. From: Bacillus subtilis RNase J1 endonuclease and 5? exonuclease activities in the turnover of ?ermC mRNA.

Northern blot analysis of (A) 3′-end-containing decay intermediates detected with (Δe) and without (stop > 2) translation, and (B) processed RNA and decay intermediates in the presence of Em-induced ribosome stalling.

Shiyi Yao, et al. RNA. 2009 December;15(12):2331-2339.
2.
FIGURE 4.

FIGURE 4. From: Bacillus subtilis RNase J1 endonuclease and 5? exonuclease activities in the turnover of ?ermC mRNA.

Deletion analysis of ΔermC mRNA. Schematic diagram of (A) wild-type (wt) ΔermC mRNA is shown at the top, with downward arrows indicating putative sites of RNase J1 endonucleolytic cleavage. Half-lives (average of at least three determinations) are shown at right, together with P-value relative to wild-type half-life. For the deletion constructs B–J, the extent of the deletion in nucleotides is indicated at the left and represented by parentheses on the schematic diagrams. Construct J contained an insertion of nucleotides 151–215 into the 5′-proximal region of construct G. Northern blot analysis of construct G half-life, in wild-type and RNase J1 conditional mutant strains (grown in the presence of IPTG), is shown at the right of the construct G schematic.

Shiyi Yao, et al. RNA. 2009 December;15(12):2331-2339.
3.
FIGURE 2.

FIGURE 2. From: Bacillus subtilis RNase J1 endonuclease and 5? exonuclease activities in the turnover of ?ermC mRNA.

Northern blot analysis of 3′-end-containing decay intermediates. (A) Steady-state pattern of 3′-end-containing decay intermediates in the wild-type (wt) and RNase J1 conditional mutant strain (J1), grown in the absence of IPTG. Blots of wild-type ΔermC RNA, NotI mutant RNA, and ClaI mutant RNA are shown, as indicated. On the left are the sizes (nucleotides) of 5′-end-labeled pSE420 TaqI DNA fragments (Brosius 1992) run in a parallel lane. On the right, the migration of full-length (FL) RNA and major decay intermediate bands are indicated. (B) High-resolution Northern blot analysis of ΔermC wild-type (Δe) and NotI construct RNA. Locations of 5′ ends of detected decay intermediates are indicated on the left. The sequencing ladder was generated from M13mp18 single-stranded DNA. Sizes of the sequencing ladder bands are indicated on the right. (C) Partial nucleotide sequence of the downstream portion of ΔermC mRNA, showing ΔermC stem–loops 1 and 2 and their predicted free energies, as well as location of NotI and ClaI mutations. Approximate 5′ ends of the two groups of decay intermediates are shown schematically below, as well as the location of the complementary 3′ probe.

Shiyi Yao, et al. RNA. 2009 December;15(12):2331-2339.
4.
FIGURE 1.

FIGURE 1. From: Bacillus subtilis RNase J1 endonuclease and 5? exonuclease activities in the turnover of ?ermC mRNA.

Diagrams of ΔermC mRNA wild type and mutants. (A) Wild-type ΔermC mRNA showing ribosome binding site (hatched box near 5′ end), site of RNase J1 cleavage upon Em-induced ribosome stalling (downward arrow), amino acids required for ribosome stalling (boxed), codons in the ribosome P site and A site when ribosome stalling occurs, transcription terminator structure (3′TT), and extent of 215-nt RNA observed upon ribosome stalling. (Note that this latter processing product was formerly called the “209-nt RNA” [Drider et al. 2002; Yao et al. 2008], since the size of full-length ermC mRNA was taken as 254 nt. We now estimate the size of full-length ΔermC mRNA to be 260 nt; hence the processed product is a 215-nt RNA.) At right are Northern blot analyses of ΔermC mRNA decay in wild-type and RNase J1 conditional mutant strains, grown in the presence of IPTG. Time indicated above each lane is minutes after rifampicin addition. The half-life value ± standard deviation, which was the average of at least three determinations, is shown below the blots. As a control for the amount of RNA loaded, blots were stripped and reprobed with a 5S rRNA probe. (B) Mutation of AU-rich ΔermC codons to GC-rich codons. (C) ΔermC mRNA with stop after codon 2 and strong stem–loop inserted after codon 11. (D) Nucleotide sequence and predicted structure of strong and moderate stem–loops inserted after codon 11. Arrowhead points to single nucleotide change that substantially weakens the structure. Free energies (kcal mol−1) of the predicted structures are at the bottom. (E) Northern blot analysis of decay of constructs with strong or moderate stem–loop insert and in the presence and absence of translation. (F) Plot of half-life data (average of three experiments) for ΔermC in wild-type strain (open circles), ΔermC in RNase J1 mutant strain (closed squares), and ΔermC SSL>11 (closed triangles).

Shiyi Yao, et al. RNA. 2009 December;15(12):2331-2339.

Supplemental Content

Recent activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...
Write to the Help Desk