Translation inhibition of the Salmonella fliC gene by the fliC 5' untranslated region, fliC coding sequences, and FlgM

The 5'-untranslated region (5'UTR) of the fliC flagellin gene of Salmonella contains sequences critical for efficient fliC mRNA translation coupled to assembly. In a previous study we used targeted mutagenesis of the 5' end of the fliC gene to isolate single base changes defective in fliC gene translation. This identified a predicted stem-loop structure, SL2, as an effector of normal fliC mRNA translation. A single base change (-38C:U) in the fliC 5'UTR resulted in a mutant that is defective in fliC mRNA translation and was chosen for this study. Motile (Mot+) revertants of the -38C:T mutant were isolated and characterized, yielding several unexpected results. Second-site suppressors that restored fliC translation and motility included mutations that disrupt a RNA duplex stem formed between RNA sequences in the fliC 5'UTR SL2 region (including a precise deletion of SL2) and bases early within the fliC-coding region. A stop codon mutation at position 80 of flgM also suppressed the -38C:T motility defect, while flgM mutants defective in anti-sigma28 activity had no effect on fliC translation. One remarkable mutation in the fliC 5'UTR (-15G:A) results in a translation defect by itself but, in combination with the -38C:U mutation, restores normal translation. These results suggests signals intrinsic to the fliC mRNA that have both positive and negative effects on fliC translation involving both RNA structure and interacting proteins.     

Regulation of rat ornithine decarboxylase mRNA translation by its 5'-untranslated region

Ornithine decarboxylase (ODC), a key enzyme in polyamine biosynthesis, is a highly inducible protein whose expression involves a complex and variable array of regulatory mechanisms. We investigated the influence of the 5'-untranslated region (5'UTR) of the rat ODC mRNA on translation of the mRNA in a cell-free system and in cultured mammalian cells. ODC mRNA containing the full-length 5'UTR was translated in reticulocyte lysates at approximately 5% of the rate of mRNA containing no ODC 5' leader sequences. The complete 5'UTR inhibited expression of a heterologous gene product, human growth hormone, to the same extent in cultured mammalian cells. Furthermore, the 5'-most 130 bases of the rat ODC 5'UTR, a conserved G/C-rich region predicted to form a stable stem-loop structure (delta G = -68 kcal/mol), repressed translation to the same extent as the entire 5'UTR, both in the lysates and in intact cells. The 3'-most 160 bases of the 5'UTR, containing a small upstream open reading frame, decreased expression by 50-65% both in vitro and in intact cells, compared with controls lacking any ODC 5'UTR sequences. Mutation of the initiation codon AUG beginning this upstream open reading frame to GCG restored expression to rates equivalent to those seen in constructions containing no ODC 5'UTR sequences. We conclude that the rat ODC mRNA 5'UTR can inhibit translation of ODC mRNA both in vitro and in vivo, and that the predicted stem-loop structure at the 5' end of the 5'UTR is both necessary and sufficient for this inhibition.     

The inhibitory effect of the 5' untranslated region of muscle acylphosphatase mRNA on protein expression is relieved during cell differentiation

Previous experiments suggested that the upstream AUG triplet present in the 5' untranslated region (UTR) of muscle acylphosphatase mRNA is involved in the regulation of protein expression. In this paper, we study the involvement of the 5'UTR secondary structure and upstream peptide on mRNA stability and protein translation. Our data, obtained using deletion and frame-shift mutants, demonstrate that the 5'UTR controls protein expression regulating translation together with mRNA stability. Furthermore, we demonstrate that the inhibitory effect of the 5'UTR of muscle acylphosphatase is relieved during the differentiation process in agreement with previous data reporting an increase of acylphosphatase content during cell differentiation. Finally, UV cross-linking experiments show that specific mRNA-binding proteins are associated with the 5'UTR of the muscle acylphosphatase mRNA.     

Translation of the leaderless Caulobacter dnaX mRNA.

The expression of the Caulobacter crescentus homolog of dnaX, which in Escherichia coli encodes both the gamma and tau subunits of the DNA polymerase III holoenzyme, is subject to cell cycle control. We present evidence that the first amino acid in the predicted DnaX protein corresponds to the first codon in the mRNA transcribed from the dnaX promoter; thus, the ribosome must recognize the mRNA at a site downstream of the start codon in an unusual but not unprecedented fashion. Inserting four bases in front of the AUG at the 5' end of dnaX mRNA abolishes translation in the correct frame. The sequence upstream of the translational start site shows little homology to the canonical Shine-Dalgarno ribosome recognition sequence, but the region downstream of the start codon is complementary to a region of 16S rRNA implicated in downstream box recognition. The region downstream of the dnaX AUG, which is important for efficient translation, exhibits homology with the corresponding region from the Caulobacter hemE gene adjacent to the replication origin. The hemE gene also appears to be translated from a leaderless mRNA. Additionally, as was found for hemE, an upstream untranslated mRNA also extends into the dnaX coding sequence. We propose that translation of leaderless mRNAs may provide a mechanism by which the ribosome can distinguish between productive and nonproductive templates.     

Genetic selection for mutations that reduce or abolish ribosomal recognition of the HIS4 translational initiator region.

A unique genetic selection was devised at the HIS4 locus to address the mechanism of translation initiation in Saccharomyces cerevisiae and to probe sequence requirements at the normal translational initiator region that might participate in ribosomal recognition of the AUG start codon. The first AUG codon at the 5' end of the HIS4 message serves as the start site for translation, and the -3 and +4 nucleotide positions flanking this AUG (AXXAUGG) correspond to a eucaryotic consensus start region. Despite this similarity, direct selection for mutations that reduce or abolish ribosomal recognition of this region does not provide any insight into the functional nature of flanking nucleotides. The only mutations identified that affected recognition of this region were alterations in the AUG start codon. Among 150 spontaneous isolates, 26 were shown to contain mutations in the AUG start codon, including all +1 changes (CUG, GUG, and UUG), all +3 changes (AUA, AUC, and AUU), and one +2 change (ACG). These seven mutations of the AUG start codon, as well as AAG and AGG constructed in vitro, were assayed for their ability to support HIS4 expression. No codon other than AUG is physiologically relevant to translation initiation at HIS4 as determined by growth tests and quantitated in his4-lacZ fusion strains. These data and analysis of other his4 alleles are consistent with a mechanism of initiation at HIS4 as proposed in the scanning model whereby the first AUG codon nearest the 5' end of the message serves as the start site for translation and points to the AUG codon in S. cerevisiae as an important component for ribosomal recognition of the initiator region.     

Conversion of the Salmonella phase 1 flagellin gene fliC to the phase 2 gene fljB on the Escherichia coli K-12 chromosome.

The Escherichia coli-Salmonella typhimurium-Salmonella abortus-equi hybrid strain EJ1420 has the two Salmonella flagellin genes fliC (antigenic determinant i) and fljB (determinant e,n,x) at the same loci as in the Salmonella strains and constitutively expresses the fliC gene because of mutations in the genes mediating phase variation. Selection for motility in semisolid medium containing anti-i flagellum serum yielded 11 motile mutants, which had the active fliC(e,n,x) and silent fljB(e,n,x) genes. Genetic analysis and Southern hybridization indicated that they had mutations only in the fliC gene, not in the fljB gene or the control elements for phase variation. Nucleotide sequence analysis of the fliC(e,n,x) genes from four representative mutants showed that the minimum 38% (565 bp) and maximum 68% (1,013 bp) sequences of the fliC(i) gene are replaced with the corresponding sequences of the fljB(e,n,x) gene. One of the conversion endpoints between the two genes lies somewhere in the 204-bp homologous sequence in the 5' constant region, and the other lies in the short homologous sequence of 6, 8, or 38 bp in the 3' constant region. The conversions include the whole central variable region of the fljB gene, resulting in fliC(e,n,x) genes with the same number of nucleotides (1,503 bp) as the fljB gene. We discuss the mechanisms for gene conversion between the two genes and also some intriguing aspects of flagellar antigenic specificities in various Salmonella serovars from the viewpoint of gene conversion.     

A snapshot of the 30S ribosomal subunit capturing mRNA via the Shine-Dalgarno interaction

In the initiation phase of bacterial translation, the 30S ribosomal subunit captures mRNA in preparation for binding with initiator tRNA. The purine-rich Shine-Dalgarno (SD) sequence, in the 5' untranslated region of the mRNA, anchors the 30S subunit near the start codon, via base pairing with an anti-SD (aSD) sequence at the 3' terminus of 16S rRNA. Here, we present the 3.3 A crystal structure of the Thermus thermophilus 30S subunit bound with an mRNA mimic. The duplex formed by the SD and aSD sequences is snugly docked in a "chamber" between the head and platform domains, demonstrating how the 30S subunit captures and stabilizes the otherwise labile SD helix. This location of the SD helix is suitable for the placement of the start codon AUG in the immediate vicinity of the mRNA channel, in agreement with reported crosslinks between the second position of the start codon and G1530 of 16S rRNA.