Determination of the Termination Efficiency of the Transcription Terminator Using Different Fluorescent Profiles in Green Fluorescent Protein Mutants

Nojima, Takahiko; Lin, Angela C; Fujii, Teruo; Endo, Itaru

doi:10.2116/analsci.21.1479

Cited by 9 publications

(5 citation statements)

References 15 publications

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“…Thus, in contrast to the Rac prophage immunity terminator, we find that λ T IMM is Rho‐independent. Our experiments show that T IMM is ~76% efficient at terminating transcription, thus similar in strength to the intrinsic terminator λ T R2 , which has a termination efficiency of ~77% (Nojima et al , ) and is located downstream of the phage replication genes (Leason and Friedman, ). In the absence of a functional T IMM terminator, transcription originating in the immunity region continues into the P L operon (Fig.…”

Section: Discussionsupporting

confidence: 63%

Elements in the λ immunity region regulate phage development: beyond the ‘Genetic Switch’

Thomason

Morrill

Murray

et al. 2019

Molecular Microbiology

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Summary Genetic elements in the bacteriophage λ immunity region contribute to stable maintenance and synchronous induction of the integrated Escherichia coli prophage. There is a bistable switch between lysogenic and lytic growth that is orchestrated by the CI and Cro repressors acting on the lytic (PL and PR) and lysogenic (PRM) promoters, referred to as the Genetic Switch. Other less well‐characterized elements in the phage immunity region include the PLIT promoter and the immunity terminator, TIMM. The PLIT promoter is repressed by the bacterial LexA protein in λ lysogens. LexA repressor, like the λ CI repressor, is inactivated during the SOS response to DNA damage, and this regulation ensures that the PLIT promoter and the lytic PL and PR promoters are synchronously activated. Proper RexA and RexB protein levels are critical for the switch from lysogeny to lytic growth. Mutation of PLIT reduces RexB levels relative to RexA, compromising cellular energetics and causing a 10‐fold reduction in lytic phage yield. The RexA and RexB proteins interact with themselves and each other in a bacterial two‐hybrid system. We also find that the transcription terminator, TIMM, is a Rho‐independent, intrinsic terminator. Inactivation of TIMM has minimal effect on λ lysogenization or prophage induction.

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Section: Discussionsupporting

confidence: 63%

Elements in the λ immunity region regulate phage development: beyond the ‘Genetic Switch’

Thomason

Morrill

Murray

et al. 2019

Molecular Microbiology

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“…The riboswitch was observed to be ‘on’ ∼35% of the time, even though no adenine was present. This ‘leakage’ rate under conditions that should turn the expression off is within the typical range of rates for similar regulatory elements such as transcription terminators (43,44). …”

Section: Resultsmentioning

confidence: 57%

Single-molecule force spectroscopy of the add adenine riboswitch relates folding to regulatory mechanism

Neupane

Foster

et al. 2011

Nucleic Acids Research

122

172

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Riboswitches regulate gene expression via ligand binding to an aptamer domain which induces conformational changes in a regulatory expression platform. By unfolding and refolding single add adenine riboswitch molecules in an optical trap, an integrated picture of the folding was developed and related to the regulatory mechanism. Force-extension curves (FECs) and constant-force folding trajectories measured on the aptamer alone revealed multiple partially-folded states, including several misfolded states not on the native folding pathway. All states were correlated to key structural components and interactions within hierarchical folding pathways. FECs of the full-length riboswitch revealed that the thermodynamically stable conformation switches upon ligand binding from a structure repressing translation to one permitting it. Along with rapid equilibration of the two structures in the absence of adenine, these results support a thermodynamically-controlled regulatory mechanism, in contrast with the kinetic control of the closely-related pbuE adenine riboswitch. Comparison of the folding of these riboswitches revealed many similarities arising from shared structural features but also essential differences related to their different regulatory mechanisms.

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“…One possibility is that transcription could be imperfectly terminated at this particular terminator and thus, the genes located downstream might be transcribed as a result of readthrough by the RNA polymerase. In this respect, previous studies have stated that termination efficiency of different terminators varies greatly both in vitro and in vivo (Reynolds et al ., 1992; Nojima et al ., 2005) based not only on the terminator structure but also upon the context of the transcription unit in which it is placed. The context affecting termination efficiency includes both early transcribed sequences (Goliger et al ., 1989; King et al ., 1996) and terminator‐distal sequences (Telesnitsky and Chamberlin, 1989).…”

Section: Discussionmentioning

confidence: 99%

“…(M) Century Plus Marker (Ambion). (Reynolds et al, 1992;Nojima et al, 2005) based not only on the terminator structure but also upon the context of the transcription unit in which it is placed. The context affecting termination efficiency includes both early transcribed sequences (Goliger et al, 1989;King et al, 1996) and terminator-distal sequences (Telesnitsky and Chamberlin, 1989).…”

Section: Discussionmentioning

confidence: 99%

Post‐transcriptional processing of the LEE4 operon in enterohaemorrhagic Escherichia coli

Lodato

Kaper²

2009

Molecular Microbiology

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Summary EnterohaemorrhagicEscherichia coli (EHEC) employs a type III secretion system (T3SS) to export translocator and effector proteins required for mucosal colonization. The T3SS is encoded in a pathogenicity island called the locus of enterocyte effacement (LEE) that is organized in five major operons, LEE1 to LEE5. LEE4 encodes a regulator of secretion (SepL), translocators (EspA, D and B), two chaperones (CesD2 and L0017), a T3SS component (EscF) and an effector protein (EspF). It was originally proposed that the esp transcript is transcribed from a promoter located at the end of sepL but other authors suggested that this transcript is the result of a posttranscriptional processing event. In this study, we established that the espADB mRNA is generated by post-transcriptional processing at the end of the sepL coding sequence. RNase E is the endonuclease involved in the cleavage, but the interaction of this enzyme with other proteins through its C-terminal half is dispensable. A putative transcription termination event in the cesD2 coding region would generate the 3Ј end of the transcript. Similar to what has been described for other processed transcripts, the cleavage of LEE4 seems a mechanism to differentially regulate SepL and Esp protein production.

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Determination of the Termination Efficiency of the Transcription Terminator Using Different Fluorescent Profiles in Green Fluorescent Protein Mutants

Cited by 9 publications

References 15 publications

Elements in the λ immunity region regulate phage development: beyond the ‘Genetic Switch’

Elements in the λ immunity region regulate phage development: beyond the ‘Genetic Switch’

Single-molecule force spectroscopy of the add adenine riboswitch relates folding to regulatory mechanism

Post‐transcriptional processing of the LEE4 operon in enterohaemorrhagic Escherichia coli

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