Natalia P. Fomenkova

Structure of ribosomal protein TL5 complexed with RNA provides new insights into the CTC family of stress proteins

The crystal structure of Thermus thermophilus ribosomal protein TL5 in complex with a fragment of Escherichia coli 5S rRNA has been determined at 2.3 A resolution. The protein consists of two domains. The structure of the N-terminal domain is close to the structure of E. coli ribosomal protein L25, but the C-terminal domain represents a new fold composed of seven beta-strands connected by long loops. TL5 binds to the RNA through its N-terminal domain, whereas the C-terminal domain is not included in this interaction. Cd(2+) ions, the presence of which improved the crystal quality significantly, bind only to the protein component of the complex and stabilize the protein molecule itself and the interactions between the two molecules in the asymmetric unit of the crystal. The TL5 sequence reveals homology to the so-called general stress protein CTC. The hydrophobic cores which stabilize both TL5 domains are highly conserved in CTC proteins. Thus, all CTC proteins may fold with a topology close to that of TL5.

A mutant form of the ribosomal protein L1 reveals conformational flexibility

The crystal structure of the mutant S179C of the ribosomal protein L1 from Thermus thermophilus has been determined at 1.9 Å resolution. The mutant molecule displays a small but significant opening of the cavity between the two domains. The domain movement seems to be facilitated by the flexibility of at least two conserved glycines. These glycines may be necessary for the larger conformational change needed for an induced fit mechanism upon binding RNA. The domain movement makes a disulfide bridge possible between the incorporated cysteines in two monomers of the mutant L1.

N-terminal domain, residues 1–91, of ribosomal protein TL5 from Thermus thermophilus binds specifically and strongly to the region of 5S rRNA containing loop E

In this work we show for the first time that the overproduced N‐terminal fragment (residues 1–91) of ribosomal protein TL5 binds specifically to 5S rRNA and that the region of this fragment containing residues 80–91 is a necessity for its RNA‐binding activity. The fragment of Escherichia coli 5S rRNA protected by TL5 against RNase A hydrolysis was isolated and sequenced. This 39 nucleotides fragment contains loop E and helices IV and V of 5S rRNA. The isolated RNA fragment forms stable complexes with TL5 and its N‐terminal domain. Crystals of TL5 in complex with the RNA fragment diffracting to 2.75 Å resolution were obtained.

Crystals of protein S6 from the 30 S ribosomal subunit of Thermus thermophilus.

Crystals of protein S6 from the small ribosomal subunit of an extreme thermophile, Thermus thermophilus, have been obtained by the hanging-drop/vapor diffusion technique using methane pentanediol as a precipitant in the presence of potassium fluoride. The crystals belong to the space group C222 with cell parameters a = 106.7, b = 52.8, c = 41.0 A. They diffract to 2.0 A resolution.

Crystallographic study of the large tryptic fragments of elongation factor G from Escherichia coli

Crystals of N- and C-terminal fragments of elongation factor G (EF-G) from Escherichia coli have been grown from the preparations obtained by limited tryptic hydrolysis. Molecular masses of these fragments are equal to about 49,000 and 25,000, respectively. In the form of an additive complex they appear to be a major part of the native spatial structure of EF-G (Alakhov et al., 1979). Crystals of N-terminal fragment belong to space group P41212 or P43212 with unit cell dimensions a = b = 76.6 A and c = 191.6 A. Crystals of C-terminal fragment belong to space group P4122 or P4322 with unit cell dimensions a = b = 77.1 A and c = 75.0 A. In both cases the assumed number of protein molecules per asymmetric part of the unit cell is one.

Crystallization and structural studies of components of the protein-synthesizing system from Thermus thermophilus

Abstract A long-term program on crystallization and structural studies of the protein synthesis machinery components from an extreme thermophile Thermus thermophilus was set up at the Institute of Protein Research (Russia) about 15 years ago. These studies have recently revealed the structures of elongation factor G, aspartyl-tRNA synthetase and ribosomal proteins S6 and L1. Different components of the protein synthesis machinery from T.thermophilus are also being investigated in other groups and many important results have been obtained recently. Here we describe only some special problems on crystal handling and non-isomorphism that have been overcome during structural studies of EF-G and ribosomal proteins in our group. This paper presents also new data on the crystallization of ribosomal proteins S7, S8, S15, L22 and leucyl-tRNA synthetase from T.thermophilus .

Crystallization and preliminary crystallographic analysis of ribosomal protein S8 from Thermus thermophilus

Crystals have been obtained for recombinant ribosomal protein S8 from Thermus thermophilus produced by Escherichia coli. The protein crystals have been grown in 40 mM potassium phosphate buffer (pH 6.0) in hanging drops equilibrated against saturated ammonium sulfate (unbuffered) with 2‐methyl‐2,4‐pentandiol (v/v). The crystals belong to the space group P41(3)212 with cell parameters a= b= 67.65 Å, c= 171.12 Å. They diffract x‐rays to 2.9 Å resolution.