Hybrid protein between ribosomal protein S16 and RimM of Escherichia coli retains the ribosome maturation function of both proteins

Abstract

The RimM protein in Escherichia coli is associated with free 30S ribosomal subunits but not with 70S ribosomes and is important for efficient maturation of the 30S subunits. A mutant lacking RimM shows a sevenfold-reduced growth rate and a reduced translational efficiency. Here we show that a double alanine-for-tyrosine substitution in RimM prevents it from associating with the 30S subunits and reduces the growth rate of E. coli approximately threefold. Several faster-growing derivatives of the rimM amino acid substitution mutant were found that contain suppressor mutations which increased the amount of the RimM protein by two different mechanisms. Most of the suppressor mutations destabilized a secondary structure in the rimM mRNA, which previously was shown to decrease the synthesis of RimM by preventing the access of the ribosomes to the translation initiation region on the rimM mRNA. Three other independently isolated suppressor mutations created a fusion between rpsP, encoding the ribosomal protein S16, and rimM on the chromosome as a result of mutations in the rpsP stop codon preceding rimM. A severalfold-higher amount of the produced hybrid S16-RimM protein in the suppressor strains than of the native-sized RimM in the original substitution mutant seems to explain the suppression. The S16-RimM protein but not any native-size ribosomal protein S16 was found both in free 30S ribosomal subunits and in translationally active 70S ribosomes of the suppressor strains. This suggests that the hybrid protein can substitute for S16, which is an essential protein probably because of its role in ribosome assembly. Thus, the S16-RimM hybrid protein seems capable of carrying out the important functions that native S16 and RimM have in ribosome biogenesis.

FIG. 1

Relative growth rates of wild-type and different mutant strains. The strains were grown in Luria broth (1), and their growth rates were normalized to that for the wild-type strain, MW100, which had a specific growth rate k (ln2/g, where g is the mass doubling time in hours) of 1.3 to 1.6 in three independent experiments. The variation between the experiments is shown as error bars.

FIG. 2

Cellular localization of RimM proteins in the wild type (A), rimM120 mutant (B), and rimM120 mutant overexpressing RimM (C). Cell extracts were fractionated by sucrose gradient centrifugation during conditions that dissociated the 70S ribosomes into 50S and 30S subunits. Selected fractions, indicated by arrows above the A₂₆₀ curve, were screened for the presence of the RimM proteins in Western blotting experiments using a polyclonal anti-RimM antiserum. The locations of the 50S and 30S ribosomal subunits are indicated below the A₂₆₀ curve.

FIG. 3

Suppressor mutations in the lower part of the mRNA secondary structure that prevents access of the ribosomes to the translational initiation region of rimM (19). The numbering is relative to the transcriptional start site of the trmD operon mRNA (6). The rpsP stop codon, SD sequence, and start codon for rimM are shaded.

FIG. 4

Amounts of RimM proteins in different strains. The amounts of the wild-type RimM and rimM120 mutant proteins in total cell extracts of strains MW100 (wt), MW136 (rimM120), JML002 (rimM120 rimM125), JML023 (rimM120 rimM129), JML024 (rimM120 rimM130), JML028 (rimM120 rimM132), and PW109 (ΔrimM102 sdr-43) were determined by using an anti-RimM antiserum in a Western blotting experiment.

FIG. 5

Production of an S16-RimM hybrid protein in the suppressor strain JML020 containing a mutation in the rpsP stop codon. The presence of the S16-RimM hybrid protein and native-sized RimM proteins was screened for in total cell extracts of strains MW100 (wt), MW136 (rimM120), PW109 (ΔrimM102 sdr-43), and JML020 (rimM120 rpsP877) by using an anti-RimM antiserum in a Western blotting experiment.

FIG. 6

Cellular localization of the S16-RimM hybrid protein. A polysome extract of strain JML020 (rimM120 rpsP877) was fractionated by sucrose gradient centrifugation, and the indicated fractions were screened for the presence of the S16-RimM hybrid protein by using an anti-RimM antiserum in a Western blotting experiment. The locations of the 50S and 30S ribosomal subunits, 70S ribosomes, and polysomes are indicated below the A₂₆₀ curve. The identity of native-sized RimM protein was determined by running a total extract of the wild-type strain MW100 and a molecular weight marker on the protein gel.

FIG. 7

Analysis of ribosomes for the presence of the r-protein S16. Acid-soluble r-proteins were subjected to immunoprecipitation with an anti-S16 antiserum after purification of 70S ribosomes and free 30S subunits from total cell extracts of strains JML020 (rimM120 rpsP877) and JML024 (rimM120 rimM130) labeled in vivo with [³⁵S]methionine (see Materials and Methods). The immunoprecipitated proteins were separated on a 10-to-17.5% gradient polyacrylamide gel containing sodium dodecyl sulfate.

FIG. 8

Location of the COOH-terminal end of the r-protein S16 in the 30S subunit of T. thermophilus. The structure of the 30S subunit is from reference and was retrieved from the Protein Data Bank (PDB no. 1FJF). Only a part of the structure is shown, in which the 16S rRNA is presented as a stick model while the indicated r-proteins are presented as space-fill models. The T. thermophilus S16 protein is 88 residues in length, but the most COOH-terminal amino acid in the determined structure is E83.