Comparative anatomy of a regulatory ribosomal protein.
Ribosomal protein L4 is a crucial folding mediator and an important architectural component of the large ribosomal subunit. Furthermore, Escherichia coli L4 produced in excess of its rRNA binding sites downregulates the transcription and translation of its own S10 operon, encoding 11 ribosomal proteins. Genetic experiments and the crystal structure of Thermotoga maritima L4 had implicated separable regions on L4 in ribosome association and expression control while RNA competition experiments and the regulatory capacity of heterologous L4 had suggested an overlap of the protein sequences involved in the two functions. We report herein that contrary to other foreign bacterial L4 proteins, L4 from T. maritima only weakly controlled expression of the S10 operon in E. coli. Also, wildtype T. maritima L4 was more weakly associated with E. coli ribosomes than with the E. coli analog. Rational mutageneses were performed to try to increase the regulatory competence of T. maritima L4. The ribosome incorporation of the mutant proteins was also investigated. Two different deletions removing T. maritima-specific sequences had little effects on regulation although one did improve ribosome association. Interestingly, a set of multiple mutations, which rendered the region around helices alpha4 and alpha5 in T. maritima L4 more E. coli-like, had no influence on the incorporation of the protein into the large ribosomal subunit but considerably improved its regulatory potential. Therefore, the area around helices alpha4 and alpha5, which is critical for the initial folding steps of the large subunit, is also a central element of autogenous control, presumably by contacting the S10 mRNA leader. Ribosome association is compounded at later stages of assembly by additional rRNA contacts through L4 areas which do not participate in regulation. Similarly, sequences outside the alpha4/alpha5 region aid expression control.
Worbs
Michael
M
Department of Biological Sciences, University of Maryland Baltimore County (UMBC), 1000 Hilltop Circle, MD, Baltimore, USA
Wahl
Markus C
MC
Lindahl
Lasse
L
Zengel
Janice M
JM
D000595:Amino Acid Sequence; D015153:Blotting, Western; D004926:Escherichia coli / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009876:Operon; D010957:Plasmids / Q000235:genetics; D011487:Protein Conformation; D012269:Ribosomal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013194:Staining and Labeling / Q000379:methods; D013329:Structure-Activity Relationship; D020124:Thermotoga maritima / Q000737:chemistry
D012269:Ribosomal Proteins; C056368:ribosomal protein L4