Ribosomal protein L22 from Thermus thermophilus: sequencing overexpression and crystallisation

Abstract

The gene for the ribosomal protein L22 from Thermus thermophilus has been sequenced and overexpressed in Escherichia coil A multiple sequence alignment was carried out for all proteins of the L22 family reported so far. The recombinant protein was purified and crystallized. The crystals belong to the space group P212~2 . with cell parameters ofa = 32.6 A, b = 66.0 A, c = 67.8 A.

The extended loops of ribosomal proteins uL4 and uL22 ofEscherichia colicontribute to ribosome assembly and protein translation

“…We therefore propose that, subsequent to formation of the initial uL4-uL24-pre23S rRNA complex, the uL4 loop and parts of p23S rRNA fold cooperatively, likely with the help of the SrmB RNA helicase, Accordingly, deletion of the uL4 loop would preclude the formation, or perhaps action, of the uL4-L24-SrmB complex, thereby changing the assembly to a slower alternate, cold sensitive path that results in functionally cold sensitive ribosomes. In contrast to uL4, the uL22 loop structure is essentially the same in crystals of both the free protein ( 58 ) and the mature ribosome ( 3 , 4 ), suggesting that uL22 modifies the rRNA structure, but is not itself significantly changed in structure.…”

Preliminary NMR studies of Thermus thermophilus ribosomal protein S19 overproduced in Escherichia coli

“…Plasmid pBR322‐tthS8 [16]for initial gene sequencing was kindly provided by Dr. V. Vysotskaya. The ribosomal protein S19 was cloned into pACA vector using the `sticky feet'‐directed mutagenesis protocol [17, 18]. The plasmid, called pTthS19, isolated from the clone with the correct gene sequence was transformed into E. coli strain B834(DE3).…”

“…The purification procedure for the ribosomal protein S19 was almost the same as for the other recently overexpressed ribosomal proteins from T. thermophilus [16, 17]with some modifications. Briefly, 3 g of wet cells were ground with alumina and resuspended in 100 mM Tris‐HCl, pH 8.1, with 800 mM NaCl, 150 mM MgCl 2 , 5 mM β‐mercaptoethanol and DNase (0.5 mg/ml final concentration).…”

Crystals of a mutant form of ribosomal protein L22 rendering bacterial ribosomes resistant to erythromycin

“…The three-amino-acid deletion was introduced into the rpl22 gene by the polymerase chain reaction (PCR) with the help of oligonucleotide primers corresponding to the region of the mutation. The plasmid pTthL22 (Davydova et al, 1995), which contains the rpl22 gene from T. thermophilus, was used as a PCR template. The resulting mutant gene was cloned into the pET 11c expression vector using BamHI and NdeI restriction sites and transformed into E. coli strain TG1.…”

“…The mutant protein was puri®ed by column chromatography similar to that in Davydova et al (1995). 3 g of wet cells were disintegrated by grinding with alumina and resuspended in 30 ml 50 mM sodium acetate pH 5.5, 150 mM MgCl 2 , 800 mM NaCl.…”