Mechanism of inhibition of bacteriophage T7 RNA polymerase by T7 lysozyme

Zhang, X; Studier, F. William

doi:10.1006/jmbi.1997.1016

Cited by 73 publications

(76 citation statements)

References 41 publications

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“…Also, a moderate inhibition of the T7 RNAP by T7 lysozyme results in a considerable increase in steady-state mRNA and in higher protein production (Figs 1b and 2). The finding that the presence of T7 lysozyme leads to a significant accumulation of GNA33 mRNA is consistent with the model presented by Zhang & Studier (1997) for the mechanism of T7 RNAP inhibition by T7 lysozyme. The authors proposed that lysozyme binds RNAP in the initiation conformation and prevents its conversion to an elongating complex, but it does not interfere with the polymerase during elongation.…”

Section: Discussionsupporting

confidence: 90%

“…The authors proposed that lysozyme binds RNAP in the initiation conformation and prevents its conversion to an elongating complex, but it does not interfere with the polymerase during elongation. It has been suggested also that lysozyme would increase pausing of the RNAP at specific sites that favour a transition in the RNAP, from the elongation to the initiation conformation (Zhang & Studier, 1997;Lyakhov et al, 1998).…”

Section: Discussionmentioning

confidence: 99%

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The signal peptide sequence of a lytic transglycosylase of Neisseria meningitidis is involved in regulation of gene expression

Serruto

Galeotti

2004

Microbiology

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The 60 nucleotides encoding the signal peptide of the Neisseria meningitidis membrane-bound lytic transglycosylase (MltA) homologue GNA33 were found to exert a negative regulatory effect on expression of GNA33 from either a T7-or a P lac -driven system in Escherichia coli. Down-regulation was observed to occur at the transcriptional/post-transcriptional level and could possibly be ascribed to the formation of a stem-loop secondary structure within the signal peptide sequence. Slowing down the transcription rate through inhibition/titration of the RNA polymerase resulted in a considerable increase in mRNA accumulation, suggesting that a better coupling of translation to transcription would impede the formation of the putative secondary structure. Screening of synonymous mutations in the signal peptide sequence that showed high-level expression of an in-frame fusion to a reporter resulted in the isolation of several deletion mutants lacking most of the sequence participating in the putative secondary structure. Interestingly, the increase in the steady-state mRNA level observed in deletion mutants was higher, reaching a 300-fold increment, than that found in substitution mutants. Our results support the hypothesis that the rate of transcription controls the formation of a secondary structure in the region of the GNA33 transcript corresponding to the signal peptide sequence and this, when formed, negatively regulates expression.

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Section: Discussionsupporting

confidence: 90%

Section: Discussionmentioning

confidence: 99%

The signal peptide sequence of a lytic transglycosylase of Neisseria meningitidis is involved in regulation of gene expression

Serruto

Galeotti

2004

Microbiology

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“…By contrast, the class II signal found in the concatemer junction (CJ) of replicating T7 DNA (Fig. 4 A) lacks an extended U-rich region, and most transcription complexes that encounter this signal pause and then slowly resume transcription (14,16). The different responses of ECs at these two signals is likely to ref lect differences in stability of the paused complexes caused by the U-rich region, which is known to decrease the stability of an RNA-DNA hybrid (27).…”

Section: Crosslinking the Rnap In The Ic Conformation Blocks Transcrimentioning

confidence: 99%

“…Class II pause͞termination signals such as PTH, the termination site for T7 RNAP found in human preparathyroid hormone gene, do not involve an apparent RNA structure, but require a conserved 8-bp sequence [(C͞U)AUCUGU(U͞A)] encoded in the DNA template (11)(12)(13)(14)(15). Although the class II signal located in the concatamer junction (CJ) of T7 DNA serves as a strong pause site that does not give rise to efficient termination, the presence of additional U residues downstream of the conserved sequence results in efficient termination (13,14,16). Termination at class I and class II signals appears to involve separate mechanisms, as mutations of the RNAP that prevent termination at class II signals do not affect termination at class I signals (9,17,18).…”

Section: Crosslink ͉ Transcriptionmentioning

confidence: 99%

Probing conformational changes in T7 RNA polymerase during initiation and termination by using engineered disulfide linkages

Temiakov²,

Anikin³

et al. 2005

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

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During the transition from an initiation complex to an elongation complex (EC), the single-subunit bacteriophage T7 RNA polymerase (RNAP) undergoes dramatic conformational changes. To explore the significance of these changes, we constructed mutant RNAPs that are able to form disulfide bonds that limit the mobility of elements that are involved in the transition (or its reversal) and examined the effects of the crosslinks on initiation and termination. A crosslink that is specific to the initiation complex conformation blocks transcription at 5-6 nt, presumably by preventing isomerization to an EC. A crosslink that is specific to the EC conformation has relatively little effect on elongation or on termination at a class I terminator (T ), which involves the formation of a stable stem-loop structure in the RNA. Crosslinked ECs also pause and resume transcription normally at a class II pause site (concatamer junction) but are deficient in termination at a class II terminator (PTH, which is found in human preparathyroid hormone gene), both of which involve a specific recognition sequence. The crosslinked amino acids in the EC lie close to the upstream end of the RNA-DNA hybrid and may prevent a movement of the polymerase that would assist in displacing or releasing RNA from a relatively unstable DNA-RNA hybrid in the paused PTH complex. crosslink ͉ transcriptionA lthough it consists of a single subunit, T7 RNA polymerase (RNAP) carries out all of the steps in the transcription cycle in the same manner as multisubunit RNAPs (1). Therefore, it provides a useful model for studies of the fundamental aspects of transcription. As is the situation with other RNAPs, T7 RNAP forms an unstable initiation complex (IC) that synthesizes and releases short abortive transcripts before it isomerizes to a stable elongation complex (EC) (2). The transition to an EC is accompanied by major structural rearrangements in the protein, resulting in the formation of elements that are important for the stability and processivity of the EC. These include a cavity that can accommodate an RNA-DNA hybrid of 8-9 bp and pores for RNA exit and substrate entry (3, 4). These features of the T7 RNAP EC are similar to those of the multisubunit RNAPs (5, 6).The structural rearrangements leading to an EC largely involve the N-terminal domain of the RNAP (3, 4). In particular, a ''core'' subdomain (residues 72-151 and 206-257) rotates and translocates 35 Å as a rigid body to allow room for the RNA-DNA hybrid. Other regions in the N-terminal domain become refolded to form an element that interacts with the RNA-DNA hybrid and participates in RNA displacement and resolution of the transcription bubble (the ''flap'' subdomain, residues 152-205). A structural element in the C-terminal domain (the specificity loop, residues 739-769) also becomes rearranged during the transition. Whereas in the IC this element is involved in promoter contacts, in the EC it becomes associated with the displaced transcript and forms part of the RNA exit pore (7, 8) (see Fig. 1).T...

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“…This effect can be measured as an apparent T7 lysozyme-induced increase in the K NTP (Huang et al 1999;Villemain and Sousa 1998). Once the polymerase has cleared the promoter, the elongation complex (EC) is generally resistant to T7 lysozyme (Zhang and Studier 1997;Kumar and Patel 1997). However, if the RNA:T7-RNAP interaction is disrupted the EC becomes sensitive to T7 lysozyme (Huang et al 1999).…”

Section: T7 T7-rnap:t7 Lysozyme Networkmentioning

confidence: 99%

Design and implementation of three incoherent feed-forward motif based biological concentration sensors

2007

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Synthetic biology is a useful tool to investigate the dynamics of small biological networks and to assess our capacity to predict their behavior from computational models. In this work we report the construction of three different synthetic networks in Escherichia coli based upon the incoherent feed-forward loop architecture. The steady state behavior of the networks was investigated experimentally and computationally under different mutational regimes in a population based assay. Our data shows that the three incoherent feed-forward networks, using three different macromolecular inhibitory elements, reproduce the behavior predicted from our computational model. We also demonstrate that specific biological motifs can be designed to generate similar behavior using different components. In addition we show how it is possible to tune the behavior of the networks in a predicable manner by applying suitable mutations to the inhibitory elements.

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Mechanism of inhibition of bacteriophage T7 RNA polymerase by T7 lysozyme

Cited by 73 publications

References 41 publications

The signal peptide sequence of a lytic transglycosylase of Neisseria meningitidis is involved in regulation of gene expression

The signal peptide sequence of a lytic transglycosylase of Neisseria meningitidis is involved in regulation of gene expression

Probing conformational changes in T7 RNA polymerase during initiation and termination by using engineered disulfide linkages

Design and implementation of three incoherent feed-forward motif based biological concentration sensors

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