Sequence-specific recognition of RNA hairpins by bacteriophage antiterminators requires a conserved arginine-rich motif

Lazinski, David W.; Grzadzielska, Elizabeth; Das, Asis

doi:10.1016/0092-8674(89)90882-9

Cited by 402 publications

(325 citation statements)

References 48 publications

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“…All but one of these were found in Gram-positive or near Gram-positive genera, the only exception being the Gram-impermeable spirochete, Borrelia burgdorferi+ An alignment of these enzymes is shown in Figure 6 and highlights two highly conserved regions, one at the immediate amino-terminus of the protein, the other at about one-third (amino acids 59-74), and a third less obvious domain about three-quarters distance (amino acids 156-159) along the protein+ A BLAST search with the second conserved domain alone revealed weak homology with a domain of the archaebacterial reverse gyrases, of the type I 59-topoisomerase family, which nick double-stranded DNA en route to the introduction of positive supercoils in the DNA (Jaxel et al+, 1999)+ The third conserved region contained an arginine rich motif resembling that found in the RNAbinding domain of proteins such as lambda N or HIV Tat proteins (Lazinski et al+, 1989)+…”

Section: S Rrna Precursors Are Incorporated Into Translating Ribosomesmentioning

confidence: 99%

Identification of the gene encoding the 5S ribosomal RNA maturase in Bacillus subtilis: Mature 5S rRNA is dispensable for ribosome function

Condon

Bréchemier-Baey

Beltchev

et al. 2001

RNA

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Over 25 years ago, Pace and coworkers described an activity called RNase M5 in Bacillus subtilis cell extracts responsible for 5S ribosomal RNA maturation (Sogin & Pace, Nature, 1974, 252:598-600). Here we show that RNase M5 is encoded by a gene of previously unknown function that is highly conserved among the low G 1 C Gram-positive bacteria. We propose that the gene be named rnmV. The rnmV gene is nonessential. B. subtilis strains lacking RNase M5 do not make mature 5S rRNA, indicating that this process is not necessary for ribosome function. 5S rRNA precursors can, however, be found in both free and translating ribosomes. In contrast to RNase E, which cleaves the Escherichia coli 5S precursor in a single-stranded region, which is then trimmed to yield mature 5S RNA, RNase M5 cleaves the B. subtilis equivalent in a double-stranded region to yield mature 5S rRNA in one step. For the most part, eubacteria contain one or the other system for 5S rRNA production, with an imperfect division along Gram-negative and Gram-positive lines. A potential correlation between the presence of RNase E or RNase M5 and the single-or double-stranded nature of the predicted cleavage sites is explored.

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Section: S Rrna Precursors Are Incorporated Into Translating Ribosomesmentioning

confidence: 99%

Identification of the gene encoding the 5S ribosomal RNA maturase in Bacillus subtilis: Mature 5S rRNA is dispensable for ribosome function

Condon

Bréchemier-Baey

Beltchev

et al. 2001

RNA

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“…Its sequence varies among relatives of A which encode distinct genomespecific N homologues (19). Chimeric nut sites that contain the same boxA but unique boxB elements permit antitermination only by the cognate N proteins (20). Hence, N might recognize boxB.…”

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confidence: 99%

“…Hence, N might recognize boxB. The specificity of hybrid N proteins suggests that a putative boxB recognition domain maps in the amino terminus of each N protein (20). This domain comprises an essential, arginine-rich motif conserved in some RNA-binding proteins (20,21).…”

mentioning

confidence: 99%

“…E. coli N100 (recA galK) was used for cloning by standard procedures. Activity of boxB mutants was assessed by galactokinase assays (20).RNAs were synthesized in vitro by transcription of linearized plasmids that contained a HindIII-boxA-boxB-EcoRI cassette or an XhoI-boxB-EcoRI cassette cloned downstream from the phage T3 or T7 promoter in standard reactions (Stratagene) with 50-500 ,tCi (1 p.Ci = 37 kBq).of [a-32P]ATP (binding studies) or [3H]UTP (competition studies). RNAs were purified by phenol/chloroform extraction and ethanol precipitation and quantitated as trichloroacetic acidprecipitable radioactivity.…”

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confidence: 99%

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Bipartite function of a small RNA hairpin in transcription antitermination in bacteriophage lambda.

Chattopadhyay

García‐Mena

DeVito

et al. 1995

Proc. Natl. Acad. Sci. U.S.A.

104

119

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Transcription of downstream genes in the early operons of phage A requires a promoter-proximal element known as nut. This site acts in cis in the form of RNA to assemble a transcription antitermination complex which is composed of A N protein and at least four host factors. The nut-site RNA contains a small stem-loop structure called boxB. Here, we show that boxB RNA binds to N protein with high affinity and specificity. While N binding is confined to the 5' subdomain of the stem-loop, specific N recognition relies on both an intact stem-loop structure and two critical nucleotides in the pentamer loop. Substitutions of these nucleotides affect both N binding and antitermination. Remarkably, substitutions of other loop nucleotides also diminish antitermination in vivo, yet they have no detectable effect on N binding in vitro. These 3' loop mutants fail to support antitermination in a minimal system with RNA polymerase (RNAP), N, and the host factor NusA. Furthermore, the ability of NusA to stimulate the formation of the RNAP-boxB-N complex is diminished with these mutants. Hence, we suggest that boxB RNA performs two critical functions in antitermination. First, boxB binds to N and secures it near RNAP to enhance their interaction, presumably by increasing the local concentration of N. Second, boxB cooperates with NusA, most likely to bring N and RNAP in close contact and transform RNAP to the termination-resistant state.The positive control of genes that facilitate the bimodal development of A and related phages in Escherichia coli depends on two distinct operon-specific antiterminators (1). The N antiterminator activates the early operons, whereas the Q antiterminator activates the late operon. Both proteins function by a common mechanism: they capture RNA polymerase (RNAP) during early phases of transcription and mask RNAP's response to the downstream terminators (2-8). However, each antiterminator recognizes the respective genetic signal and captures RNAP by distinct mechanisms. The signals for Q action span the late promoter and the early transcribed region. Q binds to a DNA sequence within the late promoter and acts upon RNAP paused at a defined site (9). Specific nucleotides in the nontemplate strand of this region interact with RNAP not only to induce pausing but also to endow upon RNAP the conformation that is essential for engagement by Q (10). In contrast, the nut site, required for N action, functions in the form of . It can facilitate the productive interaction between N and RNAP at remote sites, suggesting that nut RNA may act similarly to DNA enhancers, binding N and delivering N to RNAP through RNA looping (11). Finally, while a single host factor (NusA) appears to be sufficient for Q activity, processive antitermination by N demands three additional factors: NusB, S10 ribosomal protein (NusE), and NusG (2, 14-16).The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §173...

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“…Assembly of the antitermination complex requires a cis-acting RNA sequence called the nut site which contains two distinct sequence elements: boxB, which binds N, and boxA, which interacts with the host accessory factors NusB and S10 (Nodwell & Greenblatt 1991Franklin 1985). A variety of experiments have revealed that N binds to boxB through a short N-terminal region, while the remainder of the protein appears to interact with RNA polymerase and the host factor NusA (Greenblatt & Li 1981;Lazinski et al 1989;Whalen et al 1988). Such findings suggested to us that the N-mediated antitermination system may be modular and could be exploited to detect heterologous RNA-protein interactions.…”

Section: Introductionmentioning

confidence: 99%

A one‐hybrid system for detecting RNA–protein interactions

Wilhelm¹,

Vale²

1996

Genes to Cells

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Background : mRNA translation, stability, and localization are controlled by regulatory proteins that bind to specific RNA motifs. Since biochemical isolation of such proteins has often proven to be difficult, a genetic system for studying RNAprotein interactions would be of great utility in the identification of novel RNA binding proteins and in understanding how these proteins recognize particular RNA sequences.The bacteriophage lambda gene product N protein is a sequence specific RNA binding protein that when bound to its target sequence allows RNA polymerase to ignore transcription termination signals. The fact that the binding of N protein to RNA is directly coupled to gene expression suggests that N protein could be used to develop a general system for studying RNA-protein interactions.

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Sequence-specific recognition of RNA hairpins by bacteriophage antiterminators requires a conserved arginine-rich motif

Cited by 402 publications

References 48 publications

Identification of the gene encoding the 5S ribosomal RNA maturase in Bacillus subtilis: Mature 5S rRNA is dispensable for ribosome function

Identification of the gene encoding the 5S ribosomal RNA maturase in Bacillus subtilis: Mature 5S rRNA is dispensable for ribosome function

Bipartite function of a small RNA hairpin in transcription antitermination in bacteriophage lambda.

A one‐hybrid system for detecting RNA–protein interactions

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