Unusual oligomerization required for activity of NtrC, a bacterial enhancer-binding protein

Science

Volumn 275, Issue 5306, 1997, Pages 1658-1661

  Wyman, Claire ^b   Rombel, Irene ^a   North, Anne K ^a   Bustamante, Carlos ^c   Kustu, Sydney ^a  

^a UNIVERSITY OF CALIFORNIA   (United States)

^b ERASMUS MEDICAL CENTER   (Netherlands)

^c UNIVERSITY OF OREGON   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

ARTICLE; DNA BINDING; NONHUMAN; OLIGOMERIZATION; PRIORITY JOURNAL; PROTEIN DNA INTERACTION; SALMONELLA TYPHIMURIUM; TRANSCRIPTION INITIATION;

ADENOSINE TRIPHOSPHATASES; BACTERIAL PROTEINS; DIMERIZATION; DNA, BACTERIAL; DNA-BINDING PROTEINS; DNA-DIRECTED RNA POLYMERASES; ENHANCER ELEMENTS (GENETICS); PHOSPHORYLATION; PII NITROGEN REGULATORY PROTEINS; PROMOTER REGIONS (GENETICS); RNA POLYMERASE SIGMA 54; SALMONELLA TYPHIMURIUM; SIGMA FACTOR; TRANS-ACTIVATION (GENETICS); TRANS-ACTIVATORS; TRANSCRIPTION FACTORS; TRANSCRIPTION, GENETIC;

BACTERIA (MICROORGANISMS); EUKARYOTA; SALMONELLA TYPHIMURIUM; TYPHIMURIUM;

EID: 0030894489     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.275.5306.1658     Document Type: Article

References (47)

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14. Large oligomers were not detected in gel mobility shift assays, presumably because dimers not directly DNA bound (see text) dissociated during the course of electrophoresis (7). P-NtrC apparently formed large oligomers at the glnA enhancer in electron micrographs [W. Su, S. Porter, S. Kustu, H. Echols, Proc. Natl. Acad. Sci. U.S.A. 87, 5504 (1990)], but their size could not be reliably determined, principally because images were distorted by staining and shadowing materials. Large oligomers of P-NtrC could not be studied by scanning transmission electron microscopy, because they were obscured by the background of free protein necessary to form them (M. Simon and I. Rombel, unpublished data). Deposition of samples for SFM takes <1 min. This technique can be used to obtain topographic images of biomolecules directly without the use of external means of contrast, and three-dimensional information can be obtained without complicated image reconstruction. Although SFM images are distorted in ways that alter the dimensions of biological molecules, these distortions are constant within a set of scans made with the same sample deposition and the same tip; hence, it should be possible to determine the relative sizes of biomolecules from images collected at the same time (9).
15. Large oligomers were not detected in gel mobility shift assays, presumably because dimers not directly DNA bound (see text) dissociated during the course of electrophoresis (7). P-NtrC apparently formed large oligomers at the glnA enhancer in electron micrographs [W. Su, S. Porter, S. Kustu, H. Echols, Proc. Natl. Acad. Sci. U.S.A. 87, 5504 (1990)], but their size could not be reliably determined, principally because images were distorted by staining and shadowing materials. Large oligomers of P-NtrC could not be studied by scanning transmission electron microscopy, because they were obscured by the background of free protein necessary to form them (M. Simon and I. Rombel, unpublished data). Deposition of samples for SFM takes <1 min. This technique can be used to obtain topographic images of biomolecules directly without the use of external means of contrast, and three-dimensional information can be obtained without complicated image reconstruction. Although SFM images are distorted in ways that alter the dimensions of biological molecules, these distortions are constant within a set of scans made with the same sample deposition and the same tip; hence, it should be possible to determine the relative sizes of biomolecules from images collected at the same time (9).
16. Wild-type and mutant forms of NtrC from Salmonella typhimurium were overproduced and purified essentially as described (4, 7, 15, 18, 19). Templates 1, 2, and 3 (Fig. 2A) were prepared from plasmids pJES483 (18), pJES534 (7), and PRLM4, respectively, and were isolated from agarose gels [F. Ausubel et al., Current Protocols in Molecular Biology (Wiley, New York, 1996)]. The boxes represent 17-base pair (bp) NtrC-binding sites (5′-TGCACTAAAATGGTGCA-3′).
17. 70-holoenzyme form of RNA polymerase (260 nM final concentration; Epicenter Technologies, Madison, WI) and the 1.8-kb DNA fragment (140 nM final concentration) were combined in buffer in a volume of 10 μl and incubated for 15 min at 37°C.
18. The sizes of protein oligomers on DNA were determined using National Institutes of Health Image software. The average height and area of a manually defined object encircling the nucleoprotein complex were used to calculate a volume in arbitrary pixel units. The volume of an adjacent background region containing DNA was then subtracted to determine the volume of the protein oligomer.
19. Under conditions comparable to those used in these experiments (7), the strong enhancer is not fully occupied by tetramers of NtrC, resulting in a bimodal distribution of NtrC complexes at the enhancer (black bars in Fig. 2D). Volumes of dimers were subtracted before averaging those of tetramers by assuming that the peak corresponding to dimers had the same position and shape as that for dimers bound to the single strong site (gray bars in Fig. 2D).
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21. Y. Flashner, D. S. Weiss, J. Keener, S. Kustu, J. Mol. Biol. 249, 700 (1995); A. K. North, D. S. Weiss, H. Suzuki, Y. Flashner, S. Kustu, ibid. 260, 317 (1996).
22. 70-holoenzyme was supplied by Epicenter Technologies, Madison, WI.
23. Because we observed the larger NtrC complexes with both phosphorylating agents (12), we infer that they do not contain NtrB. The decrease in the proportion of single dimers upon phosphorylation agrees with the observation that phosphorylation increases the cooperativity of binding of two NtrC dimers to the enhancer (6, 7).
24. I. Rombel, C. Wyman, S. Kustu, unpublished data.
25. A. K. North and S. Kustu, J. Mol. Biol., in press.
26. A. K. North, I. Hwang, S. Kustu, unpublished data.
27. 3Ala to the DNA. A tetramer of the latter may have accounted for increased activity.
28. 3Ala proteins at a single strong binding site are most easily reconciled with those at the strong enhancer if one or two inactive dimers can be assimilated into active octamers but three or four cannot (I. Rombel and S. Kustu, unpublished data).
29. 54-dependent activator [reviewed in S. Hopper and A. Bock, J. Bacteriol. 177, 2798 (1995)].
30. 54-dependent activator [reviewed in S. Hopper and A. Bock, J. Bacteriol. 177, 2798 (1995)].
31. 54-dependent activator [reviewed in S. Hopper and A. Bock, J. Bacteriol. 177, 2798 (1995)].
32. 54-dependent activator [reviewed in S. Hopper and A. Bock, J. Bacteriol. 177, 2798 (1995)].
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45. D54E, in which there were too few RNA polymerase complexes to use the method above, the volume of two NtrC dimers was assumed to be twice the average volume of a single dimer on template 1. The standard deviation was that for single NtrC dimers in each experiment. The volume limit for NtrC tetramers was operationally defined as the average volume of a tetramer plus or minus one standard deviation. Larger oligomers or those carrying only a single NtrC dimer were defined as those that fell above or below this limit, respectively. This method of estimating the proportion of single dimers gave essentially the same results as that described in (14).
46. 54 [purified from S. typhimurium as described (19)] and core RNA polymerase (from Escherichia coli; kindly supplied by D. Hager and R. Burgess, University of Wisconsin, Madison) were 50 and 30 nM, respectively. Complete subunit mixing between homodimeric species of NtrC for control experiments was achieved as described (3, 7).
47. Supported by NIH grants GM17078 (C.W. and the late Harrison Echols), GM32543 (C.B.), and GM38361 (S.K). We thank K. Yamamoto for advice on eukaryotic transcriptional activation and R. Chambers and N. Pace for criticism of the manuscript.