The sequence of the distal end of the E. coli ribosomal RNA rrnE operon indicates conserved features are shared by rrn operons.

The 1440 nucleotides of the distal region of the E. coli ribosomal RNA operon found on the lambda aroE transducing phage has been sequenced. We show that the lambda aroE hybrid rrn operon ends after a solitary 5S RNA gene with rrnE distal sequence. A single terminator structure of dyad symmetry followed by a run of six T's have been identified and compared to other sequenced rrn terminator hairpins. Immediately adjacent to the hairpin is a region of interrupted but conserved sequence that is shared by rrnE, rrnB and rrnD. An open reading frame of 127 amino acids abuts the terminator structure. Another open reading frame of 147 amino acids is found on the opposite strand several hundred nucleotides downstream.     

malM, a new gene of the maltose regulon in Escherichia coli K12. I. malM is the last gene of the malK-lamB operon and encodes a periplasmic protein

The structure and expression of the distal part of the malK-lamB operon in Escherichia coli was studied. DNA sequencing was performed as far as a HinfI restriction site located 1313 base-pairs downstream from gene lamB. The open reading frame, formerly called molA, which begins 245 base-pairs downstream from gene lamB, is longer than was initially thought, and was renamed malM. It could encode a protein of 306 amino acid residues. The complete malM open reading frame was cloned under control of the tac 12 promoter. In maxicells, the resulting plasmid permitted tac12-promoted synthesis of two polypeptides, encoded by gene malM, with apparent molecular weights of 37 X 10(3) and 34.5 X 10(3). We provide strong evidence that the 34.5 X 10(3) Mr protein is derived from the 37 X 10(3) Mr protein by processing at the amino-terminal end, and that this processed form is located in the periplasmic space. We show that the chromosomal malM gene is expressed as part of the malK-lamB operon, and that its product is periplasmic. Finally, we demonstrate with nuclease S1 mapping experiments that the mRNA terminates at a typical rho-independent terminator located about 45 base-pairs beyond the end of gene malM, which is thus the last gene of the malK-lamB operon.     

Cloning and characterization of the hemA region of the Bacillus subtilis chromosome.

A 3.8-kilobase DNA fragment from Bacillus subtilis containing the hemA gene has been cloned and sequenced. Four open reading frames were identified. The first is hemA, encoding a protein of 50.8 kilodaltons. The primary defect of a B. subtilis 5-aminolevulinic acid-requiring mutant was identified as a cysteine-to-tyrosine substitution in the HemA protein. The predicted amino acid sequence of the B. subtilis HemA protein showed 34% identity with the Escherichia coli HemA protein, which is known to code for the NAD(P)H:glutamyl-tRNA reductase of the C5 pathway for 5-aminolevulinic acid synthesis. The B. subtilis HemA protein also complements the defect of an E. coli hemA mutant. The second open reading frame in the cloned fragment, called ORF2, codes for a protein of about 30 kilodaltons with unknown function. It is not the proposed hemB gene product porphobilinogen synthase. The third open reading frame is hemC, coding for porphobilinogen deaminase. The fourth open reading frame extends past the sequenced fragment and may be identical to hemD, coding for uroporphyrinogen III cosynthase. Analysis of deletion mutants of the hemA region suggests that (at least) hemA, ORF2, and hemC may be part of an operon.     

Control of phenylalanyl-tRNA synthetase genetic expression. Site-directed mutagenesis of the pheS, T operon regulatory region in vitro

Previous studies of phenylalanyl-tRNA synthetase expression in Escherichia coli strongly suggested that the pheS, T operon was regulated by a phenylalanine-mediated attenuation mechanism. To investigate the functions of the different segments composing the pheS, T attenuator site, a series of insertion, deletion and point mutations in the pheS, T leader region have been constructed in vitro on a recombinant M13 phage. The effects of these alterations on the regulation of the operon were measured after transferring each mutation onto a lambda phage carrying a pheS, T-lacZ fusion. The behaviours of the various mutants agree with the predictions of the attenuation model. The role of the antiterminator (2-3 pairing) as competitor of the terminator (3-4 pairing) is demonstrated by several mutations affecting the stability of the 2-3 base-pairing. The existence of deletions and point mutations in the 3-4 base-pairing shows that the terminator is essential for both expression level and regulation of the operon. Mutations in the translation initiation site of the leader peptide show that the expression of the leader peptide is essential for attenuation control. However, alteration of the translation initiation rate of the leader peptide derepresses the pheS, T operon, which is the opposite of what is observed with the trp operon. This difference is explained in terms of different translation initiation efficiencies of the leader peptides. Finally, insertion mutations, increasing gradually the distance between the leader peptide stop codon and the first strand of the antiterminator, derepress the pheS, T operon and show that formation of the antiterminator structure is under the control of the translation of the leader peptide.     

Non-AUG-initiated internal translation of the L* protein of Theiler's virus and importance of this protein for viral persistence

Theiler's virus is a neurotropic murine picornavirus which, depending on the strain, causes either acute encephalitis or persistent demyelinating disease. Persistent strains of Theiler's virus (such as DA) produce an 18-kDa protein called L* from an open reading frame overlapping that encoding the viral polyprotein. Neurovirulent strains (such as GDVII) are thought not to produce the L* protein, as the alternative open reading frame of these strains starts with an ACG codon instead of an AUG codon. However, we observed that both persistent and neurovirulent strain derivatives can produce two forms of the L* protein through unusual type II internal ribosome entry site-mediated translation. A full-length 18-kDa protein can be expressed from an ACG or an AUG initiation codon, whereas an N-terminally truncated 15-kDa product can be translated from a downstream AUG initiation codon. The expression of the 18-kDa form is required for efficient persistence of DA virus derivatives in the central nervous system.