Supercoiling, integration host factor, and a dual promoter system, participate in the control of the bacteriophage λ pL promoter

Giladi, Hilla; Koby, Simi; Gottesman, Max E.; Oppenheim, Amos B.

doi:10.1016/0022-2836(92)90461-r

Cited by 64 publications

(54 citation statements)

References 37 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…RNAI transcripts are control RNAs. P L2 , a minor promoter with its transcription start point located 42-bp upstream of that of P L , was also repressed by CI (42,43). The physiological significance of P L2 is not known.…”

Section: Resultsmentioning

confidence: 99%

“…Recent in vivo reporter assays on prm240, a downpromoter mutation of P RM , template showed that DNA looping can enhance P RM activation by fivefold (41). It has been proposed that the stimulating effect of DNA looping on the activation of P RM transcription is attributable to a sterically feasible interaction between the α-carboxyl terminal domain (α-CTD) of RNA polymerase at P RM and an up element located immediately rightward of O L 3 (42,43). Due to an antiparallel loop configuration, the up element is brought into proximity to contact the α-CTD.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Multilevel autoregulation of λ repressor protein CI by DNA looping in vitro

Lewis

Zurla

et al. 2011

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

View full text Add to dashboard Cite

The prophage state of bacteriophage λ is extremely stable and is maintained by a highly regulated level of λ repressor protein, CI, which represses lytic functions. CI regulates its own synthesis in a lysogen by activating and repressing its promoter, P RM . CI participates in long-range interactions involving two regions of widely separated operator sites by generating a loop in the intervening DNA. We investigated the roles of each individual site under conditions that permitted DNA loop formation by using in vitro transcription assays for the first time on supercoiled DNA that mimics in vivo situation. We confirmed that DNA loops generated by oligomerization of CI bound to its operators influence the autoactivation and autorepression of P RM regulation. We additionally report that different configurations of DNA loops are central to this regulation-one configuration further enhances autoactivation and another is essential for autorepression of P RM .activation | cooperativity | repression B acteriophage lambda (λ) of Escherichia coli can grow either in a lytic or lysogenic mode. In a lysogenic cell, the phageencoded λ repressor protein (CI) prevents lytic growth by directly repressing two promoters needed to express lytic functions, P L and P R (1-3). Each promoter is associated with a CI recognition site or operator,. O R is associated with promoter P RM (promoter for maintenance of repressor synthesis), which directs transcription of the cI gene in the prophage state (4-6). Each subsite binds a CI dimer (7).The CI protein autoregulates its synthesis. At low cellular CI concentration, CI enhances its own synthesis from P RM ; when high, CI represses P RM (1,8,9). It was originally believed that both positive and negative autoregulations are achieved exclusively by the action of CI dimers at the P RM -O R -P R sequence of the phage genome (1, 10) (Fig. 1A), based on the following observations. (i) There is a hierarchy of intrinsic binding affinities of a CI dimer to individual operators sites: (11)(12)(13)(14)(15)(16)(17); (ii) CI bound to the intrinsically weak O R 2 site is strengthened by cooperative interactions with CI bound to the stronger adjacent O R 1 site, and the ensemble of two CI dimers at the O R 1 ∼ O R 2 sites represses P R and activates P RM (13, 16) (Fig. 1A); (iii) at high CI concentrations, a CI dimer can bind to the weakest operator site, O R 3, repressing P RM (1, 2) (Fig. 1C). Incidentally, a second pair of CI dimers binds cooperatively to O L 1 ∼ O L 2, and represses P L (7).This model was recently modified on the basis of physical and genetic experiments showing that a CI tetramer cooperatively bound to O R 1 ∼ O R 2 interacts with a tetramer cooperatively bound to O L 1 ∼ O L 2, located 2.3 kbp away (18-21), resulting in the looping out of the intervening DNA, as shown by electron and atomic force microscopy (18,22,23). DNA loop was also observed by cooperative interactions of CI at sites separated by only five and six helical turns (23, 24). The kinetic and thermodynamic properties ...

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Multilevel autoregulation of λ repressor protein CI by DNA looping in vitro

Lewis

Zurla

et al. 2011

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

View full text Add to dashboard Cite

show abstract

“…A PCR DNA fragment extending from nucleotides Ϫ159 to ϩ157, relative to the LEE1 transcriptional start site, was ligated into the BamHI and EcoRI sites of plasmid pHG86 (12), which was derived from pRS415 (27), yielding pDF9. This plasmid was used to construct E. coli K-12 strain MC4100 lysogenized with a lambda phage containing the PLEE1-lacZ transcriptional fusion, as described previously (3,27).…”

Section: Methodsmentioning

confidence: 99%

Ler Is a Negative Autoregulator of theLEE1Operon in EnteropathogenicEscherichia coli

et al. 2005

View full text Add to dashboard Cite

Enteropathogenic Escherichia coli (EPEC) causes severe diarrhea in young children. Essential for colonization of the host intestine is the LEE pathogenicity island, which comprises a cluster of operons encoding a type III secretion system and related proteins. The LEE1 operon encodes Ler, which positively regulates many EPEC virulence genes in the LEE region and elsewhere in the chromosome. We found that Ler acts as a specific autorepressor of LEE1 transcription. We further show that Ler specifically binds upstream of the LEE1 operon in vivo and in vitro. A comparison of the Ler affinities to different DNA regions suggests that the autoregulation mechanism limits the steady-state level of Ler to concentrations that are just sufficient for activation of the LEE2 and LEE3 promoters and probably other LEE promoters. This mechanism may reflect the need of EPEC to balance maximizing the colonization efficiency by increasing the expression of the virulence genes and minimizing the immune response of the host by limiting their expression. In addition, we found that the autoregulation mechanism reduces the cell-to-cell variability in the levels of LEE1 expression. Our findings point to a new negative regulatory circuit that suppresses the noise and optimizes the expression levels of ler and other LEE1 genes.Colonizing enteropathogens compete with the gut flora to gain a foothold in the host tissue by expressing powerful colonization factors. However, to reduce the immune response of the host, the pathogen should minimize the expression of the colonization factors. To resolve this dilemma, pathogens evolved regulatory mechanisms that optimize the expression levels and timing, thus maintaining expression of just enough colonization factors and only when needed. Another layer of complexity is added when the colonization is dependent on the assembly of organelles like the type III secretion systems (TTSS), which are composed of ϳ30 different proteins of various relative amounts and encoded by several operons. In these cases, an orderly expression program is required for efficient assembly of the organelle.Enteropathogenic Escherichia coli (EPEC) causes severe diarrhea in young children. It employs the TTSS as a molecular syringe to inject a battery of toxic or colonization proteins into the membrane and cytoplasm of infected host cells (4). The TTSS and some of the effectors are encoded by a 35.6-kbp pathogenicity island, termed the locus for enterocyte effacement (LEE). The LEE consists of 41 genes, organized in five major operons (LEE1 to LEE5) and several additional transcriptional units (10,19). Ler, an H-NS paralog, encoded by the first gene of the LEE1 operon, is a key regulator of the LEE regulon, positively regulating expression of LEE2, LEE3, LEE4, LEE5, espG, and map (11,19,24,30). The regulation of ler (LEE1 operon) is complex and involves many factors, including H-NS, integration host factor (IHF), Fis, PerC, BipA, GrlA, GrlR, GadX, and quorum sensing (2,7,11,13,14,17,19,26,28,30,32). Most of these factors appe...

show abstract

“…For construction of the agn43-lacZ transcriptional fusion, a DNA fragment encompassing the agn43 promoter was amplified by PCR using the following primers : 5h-AGGATCCAGCAGGTATTCAAAT-GTCG-3h and 5h-CCGGAATTCGTTACTGTCTCTCTTG-TC-3h. After amplification, the DNA fragment was digested with BamHI and EcoRI and cloned into the corresponding sites of pHG86 (Giladi et al, 1992), which harbours a copy of the lacZ gene downstream of the multiple cloning site. The resultant plasmid was named pAG43p.…”

Section: Methodsmentioning

confidence: 99%