The mechanisms for regulation of ribosomal gene expression have been characterized in eukaryotes and eubacteria, but not yet in archaebacteria. We have studied the regulation of the synthesis of ribosomal proteins MvaLl, MvaL10, and MvaL12, encoded by the MvaLl operon of Methanococcus vannielii, a methanogenic archaebacterium. MvaLl, the homolog of the regulatory protein Li encoded by the Lll operon of Escherichia coli, was shown to be an autoregulator of the MvaLl operon. As in E. coli, regulation takes place at the level of translation. The target site for repression by MvaLl was localized by site-directed mutagenesis to a region within the coding sequence of the MvaLl gene commencing about 30 bases downstream of the ATG initiation codon. The MvaLl binding site on the mRNA exhibits similarity in both primary sequence and secondary structure to the Li regulatory target site ofE. coli and to the putative binding site for MvaLl on the 23S rRNA. In contrast to other regulatory systems, the putative MvaLl binding site is located in a sequence of the mRNA which is not in direct contact with the ribosome as part of the initiation complex. Furthermore, the untranslated leader sequence is not involved in the regulation. Therefore, we suggest that a novel mechanism of translational feedback regulation exists in M. vannielii.Ribosomes from different species of archaebacteria have been investigated extensively in the past, largely because they have proven to be excellent phylogenetic probes to study molecular evolution (38,39). A large amount of data is now available on ribosomal (r) proteins of archaebacteria. On the whole, the primary structures of archaebacterial r proteins are more related to their eukaryotic than to their eubacterial counterparts. In contrast, the genes for r proteins are arranged in operons as in eubacteria; even the order of the genes within the operons is similar to that found in eubacteria. Methanococcus vannielii, a methanogenic archaebacterium, contains more than 60 r proteins (49), a value intermediate between that found in eubacteria and that in eukaryotes. In general, the Methanococcus r proteins have higher molecular masses than their eubacterial homologs. As in Escherichia coli, the archaebacterial r protein equivalent to L12 is present in four copies per ribosome (7). In M. vannielii, three r protein operons have been characterized in detail: two transcription units corresponding to the E. coli spectinomycin and streptomycin operon (1,35), and the MvaLl operon (3). The MvaLl operon encodes the r proteins MvaLi, MvaL10, and MvaL12 (Fig. 1) rium cutirubrum (50) and in the sulfur-dependent thermophilic archaebacterium Sulfolobus solfataricus (51), the equivalent r protein genes are clustered in the same order as in E. coli. The regulation of the expression of many r protein operons has been investigated in E. coli (27,43). In most cases, a translational feedback regulation exists, in which certain r proteins act as autogenous repressors, controlling the expression of the operon in which they...