Tài liệu Báo cáo khóa học: The C-terminal domain of Escherichia coli Hfq increases the stability of

The C-terminal domain of Escherichia coli Hfq increases the stability

of the hexamer

Ve´ronique Arluison1

, Marc Folichon1

, Sergio Marco2

, Philippe Derreumaux3

, Olivier Pellegrini1

,

Je´roˆ me Seguin4

, Eliane Hajnsdorf1 and Philippe Regnier1

1

Institut de Biologie Physico-Chimique CNRS UPR 9073 conventionne´e avec l’universite´ Paris 7, Paris, France; 2

Institut Curie CNRS

UMR 168, Paris, France; 3

Institut de Biologie Physico-Chimique CNRS UPR 9080, Paris, France; 4

Service de Biophysique des

Fonctions Membranaires, DBJC/CEA & URA 2096 CNRS, Gif/Yvette, France

The Hfq (Host factor 1) polypeptide is a nucleic acid binding

protein involved in the synthesis of many polypeptides.

Hfq particularly affects the translation and the stability of

several RNAs. In an earlier study, the use of fold recog￾nition methods allowed us to detect a relationship between

Escherichia coli Hfq and the Sm topology. This topology

was further validated by a series of biophysical studies and

the Hfq structure was modelled on an Sm protein. Hfq forms

a b-sheet ring-shaped hexamer. As our previous study pre￾dicted a large number of alternative conformations for the

C-terminal region, we have determined whether the last 19

C-terminal residues are necessary for protein function. We

find that the C-terminal truncated protein is fully capable of

binding a polyadenylated RNA (Kd of 120 pM vs. 50 pM for

full-length Hfq). This result shows that the functional core of

E. coli Hfq resides in residues 1–70 and confirms previous

genetic studies. Using equilibrium unfolding studies, how￾ever, we find that full-length Hfq is 1.8 kcalÆmol)1 more

stable than its truncated variant. Electron microscopy ana￾lysis of both truncated and full-length proteins indicates a

structural rearrangement between the subunits upon trun￾cation. This conformational change is coupled to a reduction

in b-strand content, as determined by Fourier transform

infra-red. On the basis of these results, we propose that

the C-terminal domain could protect the interface between

the subunits and stabilize the hexameric Hfq structure. The

origin of this C-terminal domain is also discussed.

Keywords: RNA binding protein; Sm-like (L-Sm); b-topol￾ogy; urea equilibrium unfolding; electron microscopy.

Hfq (Host factor 1) of Escherichia coli is an 11 kDa

polypeptide which was originally discovered as a host factor

required for the replication of bacteriophage Qb RNA [1].

However, by inactivating of the Hfq gene, it was later

demonstrated that it is involved in a variety of other

metabolic pathways [2–4]. In particular, Hfq has been

implicated in the translation and the control of the stability

of certain mRNAs. For example, Hfq has been shown

to interfere directly with ribosome binding of the ompA

transcript, exposing the transcript to ribonucleases [5,6]. It

has also been implicated in the stimulation of the elongation

of poly(A) tails by poly(A) polymerase, leading to poly(A)-

dependent mRNA degradation [7]. Finally, it has been

shown to be involved in the translation regulation of the

rpoS transcript, encoding the rS subunit of RNA poly￾merase and, as a consequence, influences the expression of

many stationary phase genes whose transcription depends

on rS [3]. This last effect was the first cellular role observed

for Hfq and has since been the subject of much attention

because Hfq influences rpoS translation by altering the

binding of small RNAs (sRNAs) to their complementary

target sequence [8–11]. The sRNAs involved in rpoS

translation control are OxyS, DsrA, RprA. More recently,

it has been also shown that many other sRNA can interact

with Hfq, pointing to a global role of the protein in

facilitating sRNA function [12,13].

Little is known about the mechanism of Hfq action. It has

been shown to bind strongly to single-stranded RNAs that

are A and U rich. Taking into account its ability to rescue

a folding trap of a splicing defective intron [14] and its

requirement for the activity of many sRNAs [11,15], it has

been proposed to be an RNA chaperone. The interaction

between Hfq and RNA may increase the propensity of

RNA to interact with itself or other RNAs, but also its

susceptibility to nucleases or poly(A) polymerase.

Recently, the Sm-like nature of Hfq was proposed on the

basis of weak sequence similarities between the N-terminal

domain of Hfq and the Sm and Sm-like (L-Sm) proteins

of eukaryotes and archaea [11,15,16]. These proteins are

components of the spliceosome complex and are also

involved in other RNA metabolism steps [17,18]. The

relationship between Hfq and the Sm topology was further

confirmed by using fold recognition methodology and by a

series of biophysical and biochemical studies. The structure

of Hfq from E. coli was modelled on an Sm protein [16] and

Correspondence to Philippe Regnier, Institut de Biologie Physico￾Chimique CNRS UPR 9073 conventionne´e avec l’universite´ Paris 7,

13 rue P. et M. Curie, 75005 Paris, France.

Fax: + 33 1 58 41 50 20, Tel.: + 33 1 58 41 51 32,

E-mail: [email protected]

Abbreviations: ATR-FTIR, attenuated total reflectance fourier

transform infra-red; EM, electron microscopy; Hfqec, E. coli Hfq;

Hfqf, Hfq full-length; HfqNter, Hfq lacking the 19 last amino acid;

sRNAs, small RNAs.

(Received 19 December 2003, revised 2 February 2004,

accepted 6 February 2004)

Eur. J. Biochem. 271, 1258–1265 (2004)
FEBS 2004 doi:10.1111/j.1432-1033.2004.04026.x