OOP RNA, produced from multicopy plasmids, inhibits lambda cII gene expression through an RNase III-dependent mechanism.

Abstract

38000F19ure 1. 6ene5 and pr0m0ter5 1n the k c11 9ene re910n. 5h0wn 15 800 6p 0f x DNA 1n rever5e 0f the 5tandard repre5entat10n, extend1n9 fr0m p051t10n 38,800 (num6ered acc0rd1n9 t0 Dan1e15 et a1. 1983) t0 p051t10n 38,000, and 1nc1ud1n9 the cr0 and c119ene5 and the 5• end 0f the 0 9ene. 7ran5cr1pt10n fr0m pR re5u1t5 1n expre5510n 0f the cr0, c11, 0, and 0ther d0wn5tream 9ene5. 7ran5cr1pt10n fr0m pR~ 15 act1vated 6y the c11 pr0te1n and re5u1t51n an ant15en5e cr0 tran5cr1pt and expre5510n 0f the c1 and rex 9ene5 (10cated at the r19ht 0f the f19ure). 7ran5cr1pt10n fr0m P0 re5u1t51n the 77-6a5e 00P RNA tran5cr1pt, term1nat1n9 at the t 0 term1nat0r 51te. [F0r a m0re c0mp1ete d15cu5510n 0f tran5cr1pt10na1 c0ntr015 1n th15 re910n, 5ee Her5k0w1t2 and Ha9en (1980);Wu1ff and R05en6er9 (1983); Ech015 (1986)1. dem0n5trated prev10u51y. 00P RNA cann0t enc0de a 5h0rt p01ypept1de: N0 AU6 0r 6U6 c0d0n and n0 cha1n term1nat10n c0d0n ex15t 1n any frame 0f read1n9. Re5u1t5A mu1t1c0py 00P DNA p1a5m1d 1nh161t5 c11 expre5510n1n the mu1t1c0py p1a5m1d pKM2, a 398-6p ~ DNA fra9-ment c0nta1n1n9 the c11-dependent k PR~ pr0m0ter 15 fu5ed t0 the E. c011 9a1K 9ene and 90vern5 1t5 tran5cr1p-t10n {F1en et a1. 1984). 6a1act0k1na5e 5ynthe515 fr0m pKM2 15 act1vated 6y c11 pr0te1n, wh1ch may 6e pr0v1ded 6y a derepre55ed k pr0pha9e, pre5ent 1n 51n91e c0py 1n the h05t chr0m050me. H0wever, 1f a 515ter p1a5m1d that c0n-ta1n5 a 651-6p ~ p~ DNA fra9ment fu5ed t0 9a1K 15 5u6-5t1tuted f0r pKM2 1n th15 5y5tem, n0 9a1K expre5510n 15 065erved, a 5eem1n9 parad0x (7a61e 1). 7he 1ar9er PR£ DNA fra9ment a150 c0nta1n5 the 00P re910n, 1nc1ud1n9 the 00P pr0m0ter, w1th the var10u5 pr0m0ter and term1nat0r e1ement5 a11 0r1ented 1n the 5ame d1rect10n 1n the 0rder p0-t0-P~-9a1K (F19. 2). 7hat the parad0x re5u1t5 fr0m the 00P DNA 5e4uence 1n the 1ar9er PRE p1a5m1d 7a61e 1. 1nh161t10n 0f cH-9ene expre5510n 6y 00P DNA p1a5m1d5P1a5m1d5 6a1act0k1na5e act1v1ty (un1t5) pKM2 (pRE-9a1K) 700 pK0-5K650 (p0-00P; p~-9a1K) 6 pDw60 (p~-9a1K) + p8R322 (•00P) 670 pDw60 {pRn-9a1K) + pLK39 {p0-00P) 2Der1vat1ve5 0f E. c011 5tra1n Uc6183 (k1nt6 c1t5857 cr027 c11 + Pam3), c0nta1n1n9 the 1nd1cated mu1t1c0py p1a5m1d5, were 9r0wn t0 109 pha5e at 32°C and then 5h1fted t0 42°c t0 1nduce c119ene expre5510n fr0m the pr0pha9e. At 30-m1n, extract5 were made and 9a1act0k1na5e act1v1t1e5 determ1ned (F1en et a1. 1984). P1a5m1d 5tructure5 are d15p1ayed 1n F195.2 and 3.15 5u99e5ted 6y the f1nd1n9 that 9a1act0k1na5e expre5510n 6y the 0r191na1 pRE-9a1K p1a5m1d 15 a1m05t c0mp1ete1y 1nh161ted 6y 1ntr0duc1n9 a 5ec0nd mu1t1c0py p1a5m1d that c0nta1n5 a 150-6p k 00P DNA fra9ment (7a61e 1). 7h15 00P fra9ment extend5 fr0m 60 6p 6ef0re the 00P tran5cr1pt10n 5tart p01nt, thr0u9h the 00P 5e4uence, t0 13 6p 6ey0nd the 00P term1nat10n 51te (F19. 3). An 00P effect can a150 6e 065erved w1th0ut u51n9 the 9a1act0k1na5e a55ay 5y5tem: k + f0rm5 c1ear p1a4ue5 0n a var1ant 0f the E. c011 1nd1cat0r 5tra1n C600 that c0nta1n5 a der1vat1ve 0f the mu1t1c0py 00P DNA p1a5m1d pLK33. 00P RNA 15 the 1nh161t0ry a9ent1f the 00P DNA fra9men...

Rho and NusG suppress pervasive antisense transcription in Escherichia coli

“…Antisense transcripts have been identified in diverse bacteria (Georg and Hess 2011), including Bacillus subtilis (Rasmussen et al 2009;Irnov et al 2010;Nicolas et al 2012) and Escherichia coli (Peters et al 2009;Dornenburg et al 2010;Shinhara et al 2011). With the exception of a few well-studied examples (e.g., l OOP [Krinke and Wulff 1987] and E. coli GadY [Opdyke et al 2004]), bacterial antisense transcripts remain largely uncharacterized. Although some antisense transcripts have specific regulatory functions, others may result from ''transcriptional noise'' generated by nonspecific transcription initiation or weak promoters that become fixed within genes by evolutionary constraints on the coding sequence (Struhl 2007).…”

cis-Antisense RNA and Transcriptional Interference: Coupled Layers of Gene Regulation

“…Though the sequence of overlapping DNA would vary between different systems, the advantage of antisense transcription is that it allows for extensive base pairing between truncated RNA and the full-length antisense counter transcripts, hence enhancing the probability of RNA interaction. Both short antisense-RNAs [50,[52][53][54]73,74] and long antisense RNA [49,[75][76][77] have been shown to participate in antisense interaction in various bacterial systems. Therefore it is possible that the resulting sense, antisense RNA hybrid complexes between truncated and full-length RNA may be subjected to similar mechanisms of RNA degradation, transcriptional attenuation or translational inhibition [6,46].…”

“…Similarly, the 77nt OOP RNA of λ phage interacts with CII mRNA and targets it for degradation via RNaseIII-dependent cleavage, thus preventing production of the CII repressor [54]. The isiA/isiR sense-antisense pairs in Synechocystis sp PCC 6803 form a duplex, which causes degradation of the isiR mRNA, though via an unknown mechanism [64] (Figure 1c).…”

“…PCC 7120 [51] and tpxA/ratA sense-antisense pair in B. subtilis [8]. cis-antisense RNAs can exist is various sizes in naturally occurring systems, ranging between short antisense RNAs, such as the 69 nt Sar RNA of bacteriophage 22 [52], 77 nt SymR RNA of E. coli [53], 77 nt OOP RNA of bacteriophage λ [54], and 104 nt Anti-Q RNA of E. faecalis [32], and long antisense RNA's, such as the 1200 nt AmgR asRNA of S. enterica, 2 kb Anti2095 RNA of Lysteria monocytogenes [13], and 7kb MED4 RNA of Prochlorococcus spp. [55].…”

Antisense DNA and RNA: progress and prospects.

“…Recently, inhibition of expression of the X cll gene by endogenous antisense RNA has been shown to be depen dent on cleavage of the mRNA by RNase III (Krinke and Wulff 1987). RNase III cleaves both the sense and the an tisense strands in the RNA : RNA duplex.…”