A protein residing at the subunit interface of the bacterial ribosome

Abstract

Surface labeling of Escherichia coli ribosomes with the use of the tritium bombardment technique has revealed a minor unidentified ribosome-bound protein (spot Y) that is hidden in the 70S ribosome and becomes highly labeled on dissociation of the 70S ribosome into subunits. In the present work, the N-terminal sequence of the protein Y was determined and its gene was identified as yfia, an ORF located upstream the phe operon of E. coli. This 12.7-kDa protein was isolated and characterized. An affinity of the purified protein Y for the 30S subunit, but not for the 50S ribosomal subunit, was shown. The protein proved to be exposed on the surface of the 30S subunit. The attachment of the 50S subunit resulted in hiding the protein Y, thus suggesting the protein location at the subunit interface in the 70S ribosome. The protein was shown to stabilize ribosomes against dissociation. The possible role of the protein Y as ribosome association factor in translation is discussed.ribosomal proteins ͉ ribosome dissociation ͉ ribosomal surface ͉ tritium labeling T he hot tritium bombardment technique is based on replacement of hydrogen by tritium in covalent bonds of thin surface layer in macromolecules (1). The technique appears to be the most direct approach for studies of protein topography on surfaces of biological structures. Protein exposure on the surfaces of viruses (1-3), membranes (4), and ribosomes (5-9) has been determined by the use of this technique.Studying proteins of the ribosome surface with this method has lead to the finding that unidentified minor component of the ribosome corresponding to spot Y on the ribosomal protein map becomes highly exposed on dissociation of the 70S ribosome into subunits (5, 7, 9). The reassociation of ribosomal subunits by increasing Mg 2ϩ concentration resulted in reshielding of the protein Y, suggesting its location at the ribosomal subunit interface (9). In this study, the protein was identified and isolated. Its interface location was confirmed by experiments on hot tritium bombardment. The protein was shown to support the 70S ribosome in associated state at low concentration of Mg 2ϩ . Materials and MethodsMaterials. Buffer reagents were obtained from Sigma, acrylamide and methylenebisacrilamide were from Fluka, urea was from Bio-Rad, DNase I was from Serva, Butyl-Toyopearl 650S from Toyo Soda (Tokyo), and DEAE-Sepharose Fast Flow from Pharmacia. Sucrose, acetone, hydrogen peroxide, and acetic acid were from ReaKhim (Moscow, Russia).Preparation of 70S Ribosomes and Total Ribosomal Protein. Ribosomes were prepared from Escherichia coli MRE-600 cells according to Staehelin et al. (10) with modifications described in ref. 9. Salt-washed ribosomes used in binding assay, as well as ribosomal subunits, were prepared as described by Gavrilova et al. (11) with substitution of pelleting by ultracentrifugation for precipitation with (NH 4 ) 2 SO 4 at the final stage of the procedure.The standard procedure of acetic acid extraction and precipitation with acetone (12) was used to p...

Two proteins, YfiA and YhbH, associated with resting ribosomes in stationary phase Escherichia coli

“…In this paper, we call these proteins YfiA and YhbH, respectively. In the study by Agafonov et al (1999), YfiA was designated protein Y and was shown to bind to the 30S ribosomal subunit.…”

Ribosome‐associated factor Y adopts a fold resembling a double‐stranded RNA binding domain scaffold

“…Residual dipolar couplings were determined as the difference between coupling constants in the aligned and isotropic conditions, and were incorporated at the late stage of structure calculation. One-bond 1 D CH values were converted to equivalent 1 D NH values. Data for residues with 1 H-15 N heteronuclear NOE values less than 0.6 were excluded from the structure calculations.…”

“…Furthermore, pY binds to the small ribosomal subunit in an Mg 2+ -dependent manner and becomes less exposed to solvent upon association of the small and large ribosomal subunits. This suggests an intersubunit position for pY in the 70S ribosome and may explain its ribosome stabilization effect [1]. pY inhibits translation most probably by interfering with the binding of the aminoacyltRNA to the ribosomal A site [2].…”

“…Ribosome activity constitutes an important target in the control of protein expression. Protein Y, the product of yfiA gene, is a ribosome-associated protein present in E. coli and many other bacteria [1][2][3]. pY was earlier assigned to the r 54 modulation protein family based on the observation that mutations in the related downstream r 54 gene cause an increase in the level of expression from r 54 -dependent promoters [4].…”

Role of 100S ribosomes in bacterial decay period

“…Two protein factors with 40% sequence homology, HPF and YfiA (also known as RaiA or protein Y) (Agafonov et al 1999;Maki et al 2000), are involved in 100S formation and dissociation. HPF functions in the second step of the reaction, whereas YfiA functions in the dissociation of the 100S subunits (Ueta et al 2005).…”