Clustered Arginine Residues of Bacteriophage λ N Protein are Essential to Antitermination of Transcription, but Their Locale Cannot Compensate for boxB Loop Defects

Franklin, Naomi C.

doi:10.1006/jmbi.1993.1287

Cited by 53 publications

(82 citation statements)

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“…Escherichia coli N567(pBR-ptac-N*) cells (11,14), phage imm P22 Nam24, and phage Nam7am53 (13) were obtained from Naomi Franklin (University of Utah). DH5␣ cells, nut reporter cloning vector pACnutTAT13 (14), and control plasmids for the human immunodeficiency virus (HIV) Rev-Rev response element (RRE) interaction, namely, pBRN-HIVRev (17) and pAC-HIVRRE (17), were obtained from Kazuo Harada (Tokyo Gakugei University).…”

Section: Generalmentioning

confidence: 99%

“…boxBs bind noncognate N peptides poorly in vitro (2,8,44). Likewise, noncognate N-nut interactions do not function in vivo (14,28), and noncognate N proteins do not rescue N-deficient viruses (10).…”

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confidence: 99%

“…We examined the requirements of boxB recognition by P22 N using a plasmid-based reporter system that reconstitutes antitermination in Escherichia coli (14). boxBs active with P22 N were obtained from screens of two randomized loop libraries.…”

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confidence: 99%

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Bacteriophage P22 Antitermination boxB Sequence Requirements Are Complex and Overlap with Those of λ

2008

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Transcription antitermination in phages and P22 uses N proteins that bind to similar boxB RNA hairpins in regulated transcripts. In contrast to the N-boxB interaction, the P22 N-boxB interaction has not been extensively studied. A nuclear magnetic resonance structure of the P22 N peptide boxB left complex and limited mutagenesis have been reported but do not reveal a consensus sequence for boxB. We have used a plasmidbased antitermination system to screen boxBs with random loops and to test boxB mutants. We find that P22 N requires boxB to have a GNRA-like loop with no simple requirements on the remaining sequences in the loop or stem. U:A or A:U base pairs are strongly preferred adjacent to the loop and appear to modulate N binding in cooperation with the loop and distal stem. A few GNRA-like hexaloops have moderate activity. Some boxB mutants bind P22 and N, indicating that the requirements imposed on boxB by P22 N overlap those imposed by N. Point mutations can dramatically alter boxB specificity between P22 and N. A boxB specific for P22 N can be mutated to N specificity by a series of single mutations via a bifunctional intermediate, as predicted by neutral theories of evolution.and P22 share a closely related system of regulating the expression of early lytic genes by allowing transcription past terminators in the P left and P right operons (47). The recognition of the boxB RNA hairpins of nut (N-utilization) sites by the binding domains of the viral N proteins (Fig. 1A, B, and C) initiates the assembly of an antitermination complex. This complex contains N, host factor NusA, RNA polymerase, and other host factors bound to the viral nut site boxB and the nonhairpin boxA, allowing transcription to proceed through downstream transcription termination signals (32). The four wild-type nut sites of and P22 are similar in sequence but differ even between boxB left and boxB right in the same virus. Notably, both P22 boxBs have a C in the loop where both boxBs have an A, P22 boxB stems are longer than those of by 1 base pair, and P22 boxB right appears to have a noncanonical C:C base pair. boxBs bind noncognate N peptides poorly in vitro (2,8,44). Likewise, noncognate N-nut interactions do not function in vivo (14, 28), and noncognate N proteins do not rescue N-deficient viruses (10).The details of the N-boxB interaction have been revealed by extensive genetic and biochemical work (47) and by solution state nuclear magnetic resonance (NMR) structures of the arginine-rich domain of the N protein bound to boxB left (29) and boxB right (39) RNA. Genetic and biochemical studies of the P22 interaction are less complete (13, 44) but are supported by the solution state NMR structure of the arginine-rich domain of the N protein bound to P22 boxB right (5).and P22 boxBs bind their N peptides as hairpins in which 4 of 5 bases adopt a GNRA-like tetraloop structure (Fig. 2). Tetraloops are frequently found in RNAs serving structural roles as stable caps to stems and as motifs recognized by proteins; GNRA tetraloops are noted ...

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Section: Generalmentioning

confidence: 99%

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confidence: 99%

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Bacteriophage P22 Antitermination boxB Sequence Requirements Are Complex and Overlap with Those of λ

2008

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“…Reporters containing either the boxA-R17 or boxA-R17 low binding sites were constructed by altering the pAC-TAT13 reporter construct (Franklin 1993) by using partially overlapping oligonucleotides that were annealed and then extended with Klenow fragment of DNA polymerase (Madhani & Guthrie 1994). The resulting DNA fragment was digested near the ends with SalI and BamHI and then inserted into the SalI/BamHI sites of pAC-TAT13.…”

Section: Construction Of Reporter and Activator Plasmidsmentioning

confidence: 99%

“…Fusions of R17 to N protein were constructed in the expression plasmid pBR-ptac N* (Franklin 1993). Fusion of R17 to the amino terminus of N protein was done by using PCR to generate an R17 fragment that had flanking NcoI sites and a five glycine linker attached to the carboxyl terminus of R17.…”

Section: Construction Of Reporter and Activator Plasmidsmentioning

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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RNA recognition by arginine-rich peptide motifs

1998

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A ubiquitious class of RNA‐binding proteins is distinguished by an arginine‐rich motif. Such proteins function in transcription, translation, RNA trafficking, and packaging. Peptide models are derived from viral regulatory proteins, including the virulence factors Tat and Rev of mammalian immunodeficiency viruses. Structures of model peptide–RNA complexes exhibit diverse strategies of recognition based in each case on structural transitions. Induced RNA structures contain noncanonical elements such as purine–purine mismatches, base triples, and flipped bases. Such elements enlarge and extend the RNA major groove to create specific peptide‐binding pockets and surfaces. The repertoire of bound peptide structures—β‐hairpin, α‐helix, and helix–bend–helix—reflects the diversity of induced RNA architectures. This repertoire, reminiscent of primordial exon‐encoded peptides, may recapitulate early events in the transition between RNA and protein worlds. Peptide‐directed changes in modern RNA structures can provide a mechanism of signaling in higher‐order RNA–protein assemblies. © 1999 John Wiley & Sons, Inc. Biopoly 48: 167–180, 1998

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Clustered Arginine Residues of Bacteriophage λ N Protein are Essential to Antitermination of Transcription, but Their Locale Cannot Compensate for boxB Loop Defects

Cited by 53 publications

References 0 publications

Bacteriophage P22 Antitermination boxB Sequence Requirements Are Complex and Overlap with Those of λ

Bacteriophage P22 Antitermination boxB Sequence Requirements Are Complex and Overlap with Those of λ

A one‐hybrid system for detecting RNA–protein interactions

RNA recognition by arginine-rich peptide motifs

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