RESUMO
The coliphage HK022 protein Nun transcription elongation arrest factor inhibits RNA polymerase translocation. In vivo, Nun acts specifically to block transcription of the coliphage λ chromosome. Using in vitro assays, we demonstrate that Nun cross-links RNA in an RNA:DNA hybrid within a ternary elongation complex (TEC). Both the 5' and the 3' ends of the RNA cross-link Nun, implying that Nun contacts RNA polymerase both at the upstream edge of the RNA:DNA hybrid and in the vicinity of the catalytic center. This finding suggests that Nun may inhibit translocation by more than one mechanism. Transcription elongation factor GreA efficiently blocked Nun cross-linking to the 3' end of the transcript, whereas the highly homologous GreB factor did not. Surprisingly, both factors strongly suppressed Nun cross-linking to the 5' end of the RNA, suggesting that GreA and GreB can enter the RNA exit channel as well as the secondary channel, where they are known to bind. These findings extend the known action mechanism for these ubiquitous cellular factors.
Assuntos
Bacteriófago HK022/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Gênica , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Bacteriófago HK022/genética , RNA Polimerases Dirigidas por DNA/química , Regulação Viral da Expressão Gênica , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , Domínios Proteicos , RNA Viral/química , RNA Viral/genética , RNA Viral/metabolismo , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Elongação da Transcrição/química , Fatores de Elongação da Transcrição/metabolismo , Proteínas Virais/química , Proteínas Virais/genéticaRESUMO
Coliphage HK022 excludes phage λ by subverting the λ antitermination system and arresting transcription on the λ chromosome. The 12 kDa HK022 Nun protein binds to λ nascent transcript through its N-terminal Arginine Rich Motif (ARM), blocking access by λ N and arresting transcription via a C-terminal interaction with RNA polymerase. In a purified in vitro system, we recently demonstrated that Nun arrests transcription by restricting lateral movement of transcription elongation complex (TEC) along the DNA register, thereby freezing the translocation state. We will discuss some of the key experiments that led to this conclusion, as well as present additional results that further support it.
RESUMO
The Nun protein of coliphage HK022 arrests RNA polymerase (RNAP) in vivo and in vitro at pause sites distal to phage λ N-Utilization (nut) site RNA sequences. We tested the activity of Nun on ternary elongation complexes (TECs) assembled with templates lacking the λ nut sequence. We report that Nun stabilizes both translocation states of RNAP by restricting lateral movement of TEC along the DNA register. When Nun stabilized TEC in a pretranslocated register, immediately after NMP incorporation, it prevented binding of the next NTP and stimulated pyrophosphorolysis of the nascent transcript. In contrast, stabilization of TEC by Nun in a posttranslocated register allowed NTP binding and nucleotidyl transfer but inhibited pyrophosphorolysis and the next round of forward translocation. Nun binding to and action on the TEC requires a 9-bp RNA-DNA hybrid. We observed a Nun-dependent toe print upstream to the TEC. In addition, mutations in the RNAP ß' subunit near the upstream end of the transcription bubble suppress Nun binding and arrest. These results suggest that Nun interacts with RNAP near the 5' edge of the RNA-DNA hybrid. By stabilizing translocation states through restriction of TEC lateral mobility, Nun represents a novel class of transcription arrest factors.
Assuntos
RNA Polimerases Dirigidas por DNA/metabolismo , Elongação da Transcrição Genética , Fatores de Transcrição/metabolismo , Proteínas Virais/metabolismo , Bacteriófago lambda/genética , Bacteriófago lambda/metabolismo , DNA Viral/química , DNA Viral/genética , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/genética , Difosfatos/metabolismo , Modelos Genéticos , Modelos Moleculares , Mutação , Conformação de Ácido Nucleico , Nucleotídeos/genética , Nucleotídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , RNA Viral/química , RNA Viral/genética , Moldes Genéticos , Fatores de Transcrição/química , Fatores de Transcrição/genética , Proteínas Virais/química , Proteínas Virais/genéticaRESUMO
In experiments with germ free mice, free from adaptive antibodies to the bacterial virus lambda phage, titers of the virus in the circulatory system have been reported to decrease by more than 10(9)pfu within 48 h of intraperitoneal intravenous or oral administration. Based on these observations, serial passage techniques have been used to select lambda phage mutants, with 13,000-16,000-fold greater capacity to remain in the mouse circulatory system 24h after intraperitoneal injection. In these prior studies the "long-circulating" phage, designated lambdaArgo phage, had at least three mutations including one in the major phage capsid (E) protein, which resulted in the change of glutamic acid to a lysine at residue 158. In the current study, we demonstrate that this single specific substitution in the E protein is sufficient to confer the "long-circulating" phenotype. The isogenic pair of phage developed in this study consisting of the long-circulating marker-rescued lambdaArgo phage, and the parental wild type phage can be used for studies of viral recognition mechanisms of the innate immune system and for the development of more effective antibacterial therapeutic phage strains.