An electron microscopic study of the archaeal feast/famine regulatory protein

Abstract

Abstract:Particles formed by a feast/famine regulatory protein (FFRP), pot0434017 (FL11), in solution in the absence of DNA were analyzed using electron microscopy (EM). By applying conventional (i.e. dry) EM to the protein negatively stained with uranyl acetate, top views of tetrameric assemblies of dimers were obtained, where four pairs each of N-domains were extending from C-domains assembled around the centers. In cryo-EM images of the protein embedded in 3D amorphous ice, sets of four densities were arranged around ellipsoids having similar lengths for their long axes but of different lengths for their short axes. These images were interpreted as projections with different tilts of four pairs of N-domains arranged inside flat assemblies: the positively charged N-domains only were stained with ammonium molybdate, but the negatively charged C-domains were unstained and thus unobservable. Using seventeen such cryo-images, in combination with a crystal structure equivalent to an assembly of C-domains, a disk-like 3D structure was reconstructed.

Feast/famine regulatory proteins (FFRPs):Escherichia coliLrp, AsnC and related archaeal transcription factors

“…Possible directions of the DNA helix axes formed upon interaction with the FFRP assemblies are traced in (a–d). (g) An average of five conventional electron microscopy (EM) images of FL11 (Ishijima et al ., 2004), negatively stained using uranyl acetate, symmetrized beforehand by imposing a perfect four‐fold symmetry around the centre of each image and also averaged with its mirror image, producing a 4· m · m symmetry in two dimension, suggestive of 4·2·2 symmetry in three dimension.…”

“…dry; Fig. 2g) electron microscopy (EM) (Ishijima et al ., 2003, 2004a–e; Clowney et al ., 2004; Koike et al ., 2004a). No crystal structure is available of a DNA‐complex for any FFRP.…”

“…In this review, we refer to the archaeal and eubacterial proteins that are orthologous or paralogous to Escherichia coli leucine‐responsive regulatory protein (Lrp) and asparagine synthase C gene product (AsnC) as feast/famine regulatory proteins (FFRPs) (Ishijima et al ., 2003, 2004a–e; Koike et al ., 2003, 2004a; Suzuki, 2003a,b; Suzuki & Koike, 2003; Suzuki et al ., 2003a, b; Clowney et al ., 2004; Sakuma et al ., 2005; Yokoyama et al ., 2005). The term ‘feast/famine regulation’ was coined by Calvo & Matthews (1994), when summarizing functions of E. coli Lrp (Ec0932312).…”

Binding of the feast/famine regulatory protein (FFRP) FL11 (pot0434017) to DNA in the "promoter to coding" region of gene fl11

“…In fact, formation of octamers has been identified and studied using electron microscopy. 14) In an FFRP octamer the four pairs of DNA-binding domains (DBDs) will be positioned essentially flat in a 2D plane, and thus the DNA circling around these DBDs will have no superhelicity (Fig. 6b).…”

Comparison of foot-prints of FFRPs on promoters designed for autoregulation: FL10 (pot0377090, LrpA), FL11 (pot0434017), and Ss/Sa-Lrp

“…13 plus 18), thereby forming two or three helical turns of the DNA. 20) Or more generally, insertions are made by [7][8][9][10][11][12][13][14][15][16][17][18][19][20] In the upstream half of the protected region, separated from the B site with an insertion of 8 bps, symmetrically positioned are another 13 bps, GCTGATGTTATAC (bases complementary to each other are underlined): the A site (Fig. 1).…”