Independent Ligand-Induced Folding of the RNA-Binding Domain and Two Functionally Distinct Antitermination Regions in the Phage λ N Protein

Mogridge, Jeremy; Legault, Pascale; Li, Joyce; Oene, Mark D Van; Kay, Lewis E.; Greenblatt, Jack

doi:10.1016/s1097-2765(00)80027-1

Cited by 87 publications

(87 citation statements)

References 54 publications

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“…Induced-fit interactions are commonly observed in RNA-protein interactions and in arginine-rich peptide-RNA interactions in particular (35). Indeed, the arginine-rich domain of N protein is entirely disordered until it is bound to boxB (33), and the boxB loop becomes ordered upon peptide binding (4,24). The phenomenon of induced fit may increase the likelihood of sequences that can adopt more than one functional conformation, whether to signal binding, to interact with multiple partners, or to serve as intersection sequences.…”

Section: Discussionmentioning

confidence: 99%

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: Discussionmentioning

confidence: 99%

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

2008

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“…TAR RNA has been shown recently to strongly enhance the interaction between Tat and CycT1 (16). RNA-induced protein-protein interactions have been documented most clearly with the N protein, which binds to a site in the boxB RNA to mediate transcriptional antitermination (28)(29)(30)(31). The regulation of protein interactions through structural alterations in RNA could be an important mechanism for controlling the order of assembling the Tat-P-TEFb-TAR complex, both to ensure that Tat will not commit to TAR in the absence of CycT1(P-TEFb) and that P-TEFb is preferentially used at the viral promoter, because cellular genes do not express TAR RNA.…”

Section: Discussionmentioning

confidence: 99%

TAR RNA loop: A scaffold for the assembly of a regulatory switch in HIV replication

Richter

Ping²,

Rana

2002

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

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Replication of HIV requires the Tat protein, which activates elongation of RNA polymerase II transcription at the HIV-1 promoter by interacting with the cyclin T1 (CycT1) subunit of the positive transcription elongation factor complex b (P-TEFb). The transactivation domain of Tat binds directly to the CycT1 subunit of P-TEFb and induces loop sequence-specific binding of P-TEFb onto nascent HIV-1 trans-activation responsive region (TAR) RNA. We used systematic RNA-protein photocross-linking, Western blot analysis, and protein footprinting to show that residues 252-260 of CycT1 interact with one side of the TAR RNA loop and enhance interaction of Tat residue K50 to the other side of the loop. Our results show that TAR RNA provides a scaffold for two protein partners to bind and assemble a regulatory switch in HIV replication. RNA-mediated assembly of RNA-protein complexes could be a general mechanism for stable ribonucleoprotein complex formation and a key step in regulating other cellular processes and viral replication.H IV-1 encodes a transcriptional activator protein, Tat, which is expressed early in the viral life cycle and is essential for viral gene expression, replication, and pathogenesis (1-3). Tat enhances processivity of RNA polymerase II (pol II) elongation complexes that initiate in the HIV long terminal repeat region. In nuclear extracts, HIV-1 Tat associates tightly with the CDK9-containing positive transcription elongation factor complex b, P-TEFb (4-6). Recent studies indicate that Tat binds directly through its transactivation domain to the cyclin T1 (CycT1) subunit of the P-TEFb complex and induces loop sequence-specific binding of the P-TEFb complex to trans-activation responsive region (TAR) RNA (7-9).Recruitment of P-TEFb to TAR has been proposed to be both necessary and sufficient for activating transcription elongation from the HIV-1 long terminal repeat promoter (10). Neither CycT1 nor the P-TEFb complex bind TAR RNA in the absence of Tat; thus TAR binding is highly cooperative for both 9). In the C-terminal boundary of the CycT1 cyclin domain, Tat appears to contact residues that are not critical for CycT1 binding to CDK9 (8,(11)(12)(13)(14)(15). Mutagenesis studies showed that the CycT1 sequence containing amino acids 1-303 was sufficient to form complexes with Tat-TAR and CDK9 (8,(11)(12)(13)(14)(15). Recent fluorescence resonance energy-transfer studies using fluorescein-labeled TAR RNA and a rhodamine-labeled Tat protein showed that CycT1 remodels the structure of Tat to enhance its affinity for TAR RNA, and that TAR RNA further enhances interaction between Tat and CycT1 (16).The mechanism by which CycT1 induces loop sequence-specific binding of the P-TEFb complex onto nascent HIV-1 TAR RNA is not understood presently. Does CycT1 interact directly with the TAR loop or merely reorganize Tat structure to bind the loop residues? Does Tat bind TAR loop in the presence of CycT1? What regions of CycT1 and Tat interact directly with the TAR loop sequence? Does phosphorylation of P-TEFb chan...

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“…RNA-protein interactions can function cooperatively to assemble into an active complex. In ribosome assembly, protein binding induces conformational changes within the rRNA (Weeks, 1997), and in the case of the N-protein from bacteriophage , much of the protein remains unstructured until it is bound to the nut site RNA (Mogridge et al, 1998). Conformational changes within RNA-protein complexes occur to facilitate biochemical reactions.…”

Section: Discussionmentioning

confidence: 99%

Polymerization Defects within Human Telomerase Are Distinct from Telomerase RNA and TEP1 Binding

Beattie

Zhou

Robinson

et al. 2000

MBoC

114

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The minimal, active core of human telomerase is postulated to contain two components, the telomerase RNA hTER and the telomerase reverse transcriptase hTERT. The reconstitution of human telomerase activity in vitro has facilitated the identification of sequences within the telomerase RNA and the RT motifs of hTERT that are essential for telomerase activity. However, the precise role of residues outside the RT domain of hTERT is unknown. Here we have delineated several regions within hTERT that are important for telomerase catalysis, primer use, and interaction with the telomerase RNA and the telomerase-associated protein TEP1. In particular, certain deletions of the amino and carboxy terminus of hTERT that retained an interaction with telomerase RNA and TEP1 were nonetheless completely inactive in vitro and in vivo. Furthermore, hTERT truncations lacking the amino terminus that were competent to bind the telomerase RNA were severely compromised for the ability to elongate telomeric and nontelomeric primers. These results suggest that the interaction of telomerase RNA with hTERT can be functionally uncoupled from polymerization, and that there are regions outside the RT domain of hTERT that are critical for telomerase activity and primer use. These results establish that the human telomerase RT possesses unique polymerization determinants that distinguish it from other RTs. INTRODUCTIONTelomeres are the specialized nucleoprotein complexes at the physical ends of eukaryotic chromosomes (Greider and Blackburn, 1996). Telomerase is a ribonucleoprotein (RNP) enzyme that uses an internal RNA template to specifically direct telomere synthesis (Greider and Blackburn, 1985, 1987). Telomerase plays an essential role in the dynamic process of telomere length regulation in vivo by restoring telomeric sequences that are lost during semiconservative DNA replication (Watson, 1972;Olovnikov, 1973). Telomerase activity has been purified from a number of different organisms, including mammals (Greider, 1996). The telomerase RNA component (TER, telomerase RNA) has been cloned from many different species (Greider, 1996), and the secondary structure of the ciliate and mammalian telomerase RNAs is highly conserved (Romero and Blackburn, 1991;Lingner et al., 1994;McCormick-Graham and Romero, 1995;Chen et al., 2000).The first mammalian telomerase proteins (TEPs) were identified based on homology with previously cloned telomerase components from ciliates and yeast. TEP1 is a Tetrahymena p80 homolog that binds TER and is associated with telomerase activity in cell extracts (Collins et al., 1995;Harrington et al., 1997a;Nakayama et al., 1997). The mammalian telomerase reverse transcriptase (TERT) shares amino acid sequence similarity with the catalytic telomerase subunit previously identified in ciliates and yeast (Harrington et al., 1997b;Kilian et al., 1997;Lingner et al., 1997;Meyerson et al., 1997;Nakamura et al., 1997). Human TERT is a limiting component for telomerase activity: the hTERT mRNA is often up-regulated in cells containi...

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Independent Ligand-Induced Folding of the RNA-Binding Domain and Two Functionally Distinct Antitermination Regions in the Phage λ N Protein

Cited by 87 publications

References 54 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

TAR RNA loop: A scaffold for the assembly of a regulatory switch in HIV replication

Polymerization Defects within Human Telomerase Are Distinct from Telomerase RNA and TEP1 Binding

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