CHAPTER 20
Overproduction, Purification and Structural Studies
on the Zn Containing S14 Ribosomal Protein
from Thermus thermophilus
P. TSIBOLI i , D. TRIANTAFILLIDOU i , F. LEONTIADOU i , M. SIMITSOPOULOUI,
K. ANAGNOSTOPOULOS 1 , F. FRANCESCHI 2 and T. CHOLI-PAPADOPOULOU i
Introduction
Ribosomes are complexes of ribosomal RNAs and proteins. As more primary
structures of ribosomal proteins have been compiled amino acid motifs associated with nucleic acid binding have been identified in ribosomal proteins (Wool
et al. 1995). Furthermore, the cloning and overexpression of ribosomal proteins
has resulted in the elucidation of the motifs or domains implicated in rRNA binding (Ramakrishnan et al. 1995). For example the Bacillus stearothermophiius Ll1
was recently proven to bind rRNA through a helix-tum-helix domain identical to
that of the "homeodomain" regulatory proteins (Xing et al. 1997).
One motif of special interest is the zinc-finger, which is formed by a central
zinc ion co-ordinated tetrahedrally by four amino acid residues, which are either
cysteines or histidines (Kohn et al. 1997). The zinc-finger motifs in ribosomal
proteins are primarily of the C4 variety, such as those of the rat ribosomal proteins S27 and S29 (Wool et ai. 1995). Rat ribosomes have been proven to contain
zinc but there has been no evidence so far that the zinc-finger motifs of the rat
ribosomal proteins bind zinc (Chan et al. 1993). Rat S29 is related to the family
of procaryotic S14 ribosomal proteins.
Not all members of the S14 family share the zinc-finger motif (Wool et ai.
1995; Herfurth et al. 1994; Tsiboli and Choli 1994). Cross-linking studies have
indicated an interaction of the amino-terminal domain of the S14 protein of
Bacillus stearothermophilus (Urlaub et al. 1995) primarily with the 990-1045
domain of the 16S rRNA, which is highly conserved in all organisms. Although it
has not been proven, it is plausible that a zinc-finger could contribute to the
interaction of the S14 family members with the rRNA.
In this article we present our results on the cloning, overexpression and purification of the Thermus thermophiius S14 protein and we discuss the problems
encountered at every purification step as well as the rationale for the structural
studies on the overexpressed protein.
I Laboratory of Biochemistry, School of Chemistry, Aristotle University of Thessaloniki, Thessaloniki
54006, Greece.
2 Max-Planck Institut for Molecular Genetics,AG Ribosomen, Berlin 14195, Germany.
Overproduction, Purification and Structural Studies
on the Zn Containing S14 Ribosomal Protein
from Thermus thermophilus
P. TSIBOLI i , D. TRIANTAFILLIDOU i , F. LEONTIADOU i , M. SIMITSOPOULOUI,
K. ANAGNOSTOPOULOS 1 , F. FRANCESCHI 2 and T. CHOLI-PAPADOPOULOU i
Introduction
Ribosomes are complexes of ribosomal RNAs and proteins. As more primary
structures of ribosomal proteins have been compiled amino acid motifs associated with nucleic acid binding have been identified in ribosomal proteins (Wool
et al. 1995). Furthermore, the cloning and overexpression of ribosomal proteins
has resulted in the elucidation of the motifs or domains implicated in rRNA binding (Ramakrishnan et al. 1995). For example the Bacillus stearothermophiius Ll1
was recently proven to bind rRNA through a helix-tum-helix domain identical to
that of the "homeodomain" regulatory proteins (Xing et al. 1997).
One motif of special interest is the zinc-finger, which is formed by a central
zinc ion co-ordinated tetrahedrally by four amino acid residues, which are either
cysteines or histidines (Kohn et al. 1997). The zinc-finger motifs in ribosomal
proteins are primarily of the C4 variety, such as those of the rat ribosomal proteins S27 and S29 (Wool et ai. 1995). Rat ribosomes have been proven to contain
zinc but there has been no evidence so far that the zinc-finger motifs of the rat
ribosomal proteins bind zinc (Chan et al. 1993). Rat S29 is related to the family
of procaryotic S14 ribosomal proteins.
Not all members of the S14 family share the zinc-finger motif (Wool et ai.
1995; Herfurth et al. 1994; Tsiboli and Choli 1994). Cross-linking studies have
indicated an interaction of the amino-terminal domain of the S14 protein of
Bacillus stearothermophilus (Urlaub et al. 1995) primarily with the 990-1045
domain of the 16S rRNA, which is highly conserved in all organisms. Although it
has not been proven, it is plausible that a zinc-finger could contribute to the
interaction of the S14 family members with the rRNA.
In this article we present our results on the cloning, overexpression and purification of the Thermus thermophiius S14 protein and we discuss the problems
encountered at every purification step as well as the rationale for the structural
studies on the overexpressed protein.
I Laboratory of Biochemistry, School of Chemistry, Aristotle University of Thessaloniki, Thessaloniki
54006, Greece.
2 Max-Planck Institut for Molecular Genetics,AG Ribosomen, Berlin 14195, Germany.