Ends of the line for tmRNA-SmpB

Abstract

Genes for the RNA tmRNA and protein SmpB, partners in the trans-translation process that rescues stalled ribosomes, have previously been found in all bacteria and some organelles. During a major update of The tmRNA Website (relocated to http://bioinformatics.sandia.gov/tmrna), including addition of an SmpB sequence database, we found some bacteria that lack functionally significant regions of SmpB. Three groups with reduced genomes have lost the central loop of SmpB, which is thought to improve alanylation and EF-Tu activation: Carsonella, Hodgkinia, and the hemoplasmas (hemotropic Mycoplasma). Carsonella has also lost the SmpB C-terminal tail, thought to stimulate the decoding center of the ribosome. We validate recent identification of tmRNA homologs in oomycete mitochondria by finding partner genes from oomycete nuclei that target SmpB to the mitochondrion. We have moreover identified through exhaustive search a small number of complete, but often highly derived, bacterial genomes that appear to lack a functional copy of either the tmRNA or SmpB gene (but not both). One Carsonella isolate exhibits complete degradation of the tmRNA gene sequence yet its smpB shows no evidence for relaxed selective constraint, relative to other genes in the genome. After loss of the SmpB central loop in the hemoplasmas, one subclade apparently lost tmRNA. Carsonella also exhibits gene overlap such that tmRNA maturation should produce a non-stop smpB mRNA. At least some of the tmRNA/SmpB-deficient strains appear to further lack the ArfA and ArfB backup systems for ribosome rescue. The most frequent neighbors of smpB are the tmRNA gene, a ratA/rnfH unit, and the gene for RNaseR, a known physical and functional partner of tmRNA-SmpB.

Keywords: Carsonella; Mycoplasma; SmpB; tmRNA; trans-translation.

Figure 1

smpB gene neighborhoods. Each neighborhood (n = 2012) in our bacterial complete genome set was taken as the 11-gene window centered at smpB. (A) Frequent neighbors. The tmRNA gene (the only RNA gene encountered) and Pfam families present in more than 200 smpB neighborhoods are listed with a representative annotation for the instances of each family. (B) Clusters. Each neighborhood was summarized as a cluster, considering only the families of (A) (note the more specific gene annotations there). The top clusters are shown with color coding of common subclusters.

Figure 2

Carsonella smpB-ssrA: pseudogenization, neighbor gene overlap, and comparative detection of the tag reading frame. In strain PC, the three main ssrA conserved regions, at the 5′ and 3′ termini and at the tag reading frame, have all suffered so many nucleotide changes as to be unrecognizable, yet the region is largely still present. The smpB CDS (blue) extends into ssrA (expected to produce non-stop smpB mRNAs) or the ssrA pseudogene in four cases. In the HC/HT lineage, a small deletion has caused ssrA to overlap with its downstream and oppositely-oriented neighboring tRNA^Phe gene changing the last tmRNA acceptor stem (P1) nucleotide from C to U, which apparently led to a compensating G to A mutation at the first P1 nucleotide. The tag reading frame has now been determined by comparative analysis as the most conserved reading frame in ssrA, that also shares some amino acid similarity to other tag sequences. Carsonella SmpB lacks the central loop (not shown here) and the C-terminal tail, which in Thermus is a 25-residue segment following β7. The C-terminus of SmpB does extend variably beyond β7 with apparently random amino acid sequence that depends on the extent of intrusion into ssrA, but these extensions are not as long as for normal SmpBs and they do not thread into the α helix model (Kelley and Sternberg, 2009).

Figure 3

smpB and ssrA in hemoplasmas. The hemoplasmas have lost the SmpB central loop and for the suis subclade we cannot find the tmRNA gene. Genomes of 54 Mycoplasma strains were aligned using Mugsy (Angiuoli and Salzberg, 2011), yielding only the rRNA operon region as alignable for all strains; this was trimmed to 1679 bp using GBlocks requiring at least half the taxa per column (Castresana, 2000), then a maximum likelihood tree was prepared using a GTR+Γ model and autoFC bootstopping in RAxML 7.2.8 (Stamatakis, 2006). The hemoplasma clade and phylogenetic surroundings agree with recent 32-protein and 16S rRNA phylogenies (Guimaraes et al., 2014).