Kinetic measurements of Escherichia coli RNA polymerase association with bacteriophage T7 early promoters.

Dayton, C J; Prosen, Dennis E.; Parker, Keith L.; Cech, Carol L.

doi:10.1016/s0021-9258(17)43453-3

Cited by 37 publications

(22 citation statements)

References 37 publications

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“…Using the apparent on‐rate ( k 21 from fast State II to slow State I) and off‐rate ( k 12 from slow State I to fast State II) measured from SMT and an average concentration of nonspecific chromosomal DNA‐binding sites at ~5 mM under our growth condition (i.e., average two chromosomes per cell, with each chromosome having ~4 × 10 6 nucleotides, representing a total number of ~8 × 10 6 nonspecific binding sites in a total volume of ~2 fl), we estimated that the in vivo nonspecific binding affinity K d of HUα‐PAmCherry to DNA was ~4 mM and the association rate constant was ~2 × 10 3 M −1 s −1 (Supporting Table ). Both constants were two to three orders of magnitude lower than other specific DNA‐ or RNA‐binding proteins (Dayton et al ., 1984; Fei et al., 2015). Taken together, these results suggest that HU primarily interacts with chromosomal DNA in a weak and transitory manner (Dame et al ., 2013; Sarkar et al ., 2007).…”

Section: Resultsmentioning

confidence: 99%

Single‐molecule tracking reveals that the nucleoid‐associated protein HU plays a dual role in maintaining proper nucleoid volume through differential interactions with chromosomal DNA

Bettridge

Verma

Weng

et al. 2020

Molecular Microbiology

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HU (Histone‐like protein from Escherichia coli strain U93) is the most conserved nucleoid‐associated protein in eubacteria, but how it impacts global chromosome organization is poorly understood. Using single‐molecule tracking, we demonstrate that HU exhibits nonspecific, weak, and transitory interactions with the chromosomal DNA. These interactions are largely mediated by three conserved, surface‐exposed lysine residues (triK), which were previously shown to be responsible for nonspecific binding to DNA. The loss of these weak, transitory interactions in a HUα(triKA) mutant results in an over‐condensed and mis‐segregated nucleoid. Mutating a conserved proline residue (P63A) in the HUα subunit, deleting the HUβ subunit, or deleting nucleoid‐associated naRNAs, each previously implicated in HU’s high‐affinity binding to kinked or cruciform DNA, leads to less dramatically altered interacting dynamics of HU compared to the HUα(triKA) mutant, but highly expanded nucleoids. Our results suggest HU plays a dual role in maintaining proper nucleoid volume through its differential interactions with chromosomal DNA. On the one hand, HU compacts the nucleoid through specific DNA structure‐binding interactions. On the other hand, it decondenses the nucleoid through many nonspecific, weak, and transitory interactions with the bulk chromosome. Such dynamic interactions may contribute to the viscoelastic properties and fluidity of the bacterial nucleoid to facilitate proper chromosome functions.

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Section: Resultsmentioning

confidence: 99%

Single‐molecule tracking reveals that the nucleoid‐associated protein HU plays a dual role in maintaining proper nucleoid volume through differential interactions with chromosomal DNA

Bettridge

Verma

Weng

et al. 2020

Molecular Microbiology

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“…It has been shown that the rate of isomerization from a closed complex of promoter-RNA polymerase to an open active complex can be evaluated by using the abortive initiation reaction (McClure, 1980). The rates of open complex formation have been reported for about 25 promoters (McClure, 1980;Dayton et al, 1984;Hawley & McClure, 1980Stefano & Gralla, 1982;Malan et al, 1984;Shih & Gussin, 1983;Simons et al, 1983; Mulligan et al, 1985) and can be used for examining the correlation between the stability of the promoter region and the rate of open complex formation. Because the kinetic measurements of the rate constants were performed at 37 °C, we have used for this analysis free energy values corrected to 37 °C according to the data reported by Breslauer et al (1986).…”

Section: Resultsmentioning

confidence: 99%

“…This model predicts that, if k2 is almost constant, then as the free energy of transition from helix to coil decreases (helix instability increases) k{ increases. We have used a database of 25 promoter sequences for which the rates of open complex formation have been measured (McClure, 1980;Dayton et al, 1984;Hawley & McClure, 1980Stefano & Grala, 1982;Malan et al, 1984;Shih & Gussin, 1983;Simons et al, 1983;Mulligan et al, 1985) to calculate the correlation coefficient between the rate of open complex formation, k{, and e^G/RT of short blocks within the promoter region. No significant correlation has been found in either the -35 or the -10 consensus regions.…”

Section: Discussionmentioning

confidence: 99%

Helix stability in prokaryotic promoter regions

Margalit¹,

Shapiro²,

Nussinov³

et al. 1988

Biochemistry

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Prokaryotic promoters have been extensively studied to relate sequence features to promoter function. Here we examine the relationship between double-helix stability and promoter activity. The double-helix stability is evaluated from sequence data by free energy computation, based on reported values of dinucleotide free energies for strand separation. For a collection of 168 promoters, we find that within a 500-nucleotide span around the transcription initiation site the -10 region is the least stable. There is no correlation between the free energies and the rates of RNA polymerase-promoter open complex formation measured for 25 promoters. We also compare the free energies of 121 promoter mutations across the -35 and -10 consensus regions with the free energies of the corresponding wild-type sequences. These pairwise mutant-wild-type comparisons provide a particularly good test since the examined sequences differ only in one nucleotide so that all other sequence-dependent effects remain the same. About 80% of the mutations in the -10 region that show increased/reduced promoter activity are less/more stable than the wild types. The observed high free energy peak and the mutation data strongly support the conjecture that the instability, or melting properties, of the -10 region plays a significant role in promoter function.

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“…Real-time fluorescence assays eliminate the need for a competitor. Thus, they are particularly suited to investigating the reversible processes where the addition of competitors such as heparin can destabilize RPo by binding directly to intermediates [40] or eliminating the population of RPo before it can be observed.…”

Section: Kinetic Characterization Of Mtb Rpo Formationmentioning

confidence: 99%

Transcription initiation in mycobacteria: a biophysical perspective

2019

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Recent biophysical studies of mycobacterial transcription have shed new light on this fundamental process in a group of bacteria that includes deadly pathogens such as Mycobacterium tuberculosis (Mtb), Mycobacterium abscessus (Mab), Mycobacterium leprae (Mlp), as well as the nonpathogenic Mycobacterium smegmatis (Msm). Most of the research has focused on Mtb, the causative agent of tuberculosis (TB), which remains one of the top ten causes of death globally. The enzyme RNA polymerase (RNAP) is responsible for all bacterial transcription and is a target for one of the crucial antibiotics used for TB treatment, rifampicin (Rif). Here, we summarize recent biophysical studies of mycobacterial RNAP that have advanced our understanding of the basic process of transcription, have revealed novel paradigms for regulation, and thus have provided critical information required for developing new antibiotics against this deadly disease.

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Kinetic measurements of Escherichia coli RNA polymerase association with bacteriophage T7 early promoters.

Cited by 37 publications

References 37 publications

Single‐molecule tracking reveals that the nucleoid‐associated protein HU plays a dual role in maintaining proper nucleoid volume through differential interactions with chromosomal DNA

Single‐molecule tracking reveals that the nucleoid‐associated protein HU plays a dual role in maintaining proper nucleoid volume through differential interactions with chromosomal DNA

Helix stability in prokaryotic promoter regions

Transcription initiation in mycobacteria: a biophysical perspective

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