Tag Archives: E.coli

On Retrons

the secondary structure of msDNA Ec73. The 76 nt RNA (in box), is joined to a 73nt ssDNA. Note the 2'-5 phosphodiester bond connecting the two molecules at the branching guanosine.

Retrons are an understudied type of prokaryotic retroelement responsible for the synthesis of an enigmatic species of small extra-chromosomal satellite DNA termed multicopy single-stranded DNA (msDNA). msDNAs are actually composed of both a single-stranded (ss) DNA and a ssRNA. The 5′ end of the msDNA is covalently bonded to an internal guanosine residue of the msRNA by a unique 2′-5′ phosphodiester bond, whilst the 3′ ends of the molecules are joined by a small stretch of base-pairing. msDNAs are therefore a sort of looped hybrid molecule, but extensive internal base pairing creates various stem-loop/hairpin secondary structures (see figure). The retron, (ie. the genetic loci encoding the msRNA and msDNA molecules (msr and msd) and the gene encoding the reverse transcriptase (ret) responsible for the synthesis of msDNA) is transcribed as an operon.

Retrons are present in a wide variety of eubacterial, and some archaeal, genomes. A recent study identified 97 different retron-like reverse transcriptase genes within bacteria, however their distribution is sporadic. For instance, seven distinct retron elements have been found amongst E. coli strains, but only 15% of natural E. coli isolates produce msDNAs. Based on their sporadic occurrence and analysis of codon usage, retrons have been suggested to be a recent addition to the E. coli genome.

A major exception to the sporadic distribution found in most bacteria is within the myxobacteria, where all ten genera include msDNA-producing species. Myxobacterial retrons form a phylogenetically related group. These features, as well as sequence divergence, suggest that the common ancestor of the extant myxobacteria contained a retron as much as 150 million years ago, which has been vertically transmitted.

Retrons have not been shown to be mobile genetic elements, although the presence of reverse transcriptase does suggest this possibility. A clue to their propagation is the association of many of them with prophage sequences, suggesting their spread could be associated with bacteriophage. However, as with many observations about retrons, there are plenty of exceptions.

Organisation of a retron operon. note the inverse orientations and short overlap of msr and msd.

msDNA is essentially a cDNA produced from a short region of an mRNA template. During msDNA synthesis, an RNA template derived from the operon mRNA and composed of msr and msd, is folded into a specific secondary structure due to flanking inverted repeat sequences. The msd sequence is then reverse transcribed by the retron reverse transcriptase, using the 2’OH group of the ‘branching’ guanosine residue as a primer. The lagging RNA template strand is then degraded by RNaseH activity (probably host cell derived), leaving the msDNA covalently bonded at it’s 5′ end and base paired to the msRNA at their 3′ ends.

No function has been unequivocally attributed to msDNA. Mutating retron ret genes to prevent synthesis of E. coli or myxococcal msDNAs produces no detectable effects. Overexpression of certain E. coli msDNAs has been shown to increase mutation rate. msDNAs generally form hairpin structures by complementary base pairing of inverted repeat sequences (see figure). However, in many msDNA hairpins the base pairing is imperfect. It appears that the overexpression associated mutation rate phenotype is due to mismatch containing msDNAs sequestering the mismatch repair enzyme MutS. Overexpression of msDNAs without mismatch-containing hairpins does not cause similar effects. It is possible that msDNA could be regulating MutS availability by this titration mechanism in normal conditions or as part of a stress response. However, the overexpression experiments lead to msDNA concentrations far beyond normal physiological levels, so can yield no more than a hint of normal function.

In conclusion, the lacunae in our understanding of retrons and msDNA, are far more striking than the known facts. Are retrons parasitic elements? or do msDNAs have physiological roles in their host cells? Are retrons mobile elements? Just what does msDNA do? Judging from the literature, interest in retrons peaked around 1990, and recent years have been very fallow. I do hope that funding agencies and researchers keep pursuing the answers to these questions and don’t let them remain as an interesting oddity in the literature.

Lampson, B., Inouye, M., & Inouye, S. (2005). Retrons, msDNA, and the bacterial genome Cytogenetic and Genome Research, 110 (1-4), 491-499 DOI: 10.1159/000084982

Simon, D., & Zimmerly, S. (2008). A diversity of uncharacterized reverse transcriptases in bacteria Nucleic Acids Research, 36 (22), 7219-7229 DOI: 10.1093/nar/gkn867

A modified ribosome mediates stress in E.coli.

Vesper, O., Amitai, S., Belitsky, M., Byrgazov, K., Kaberdina, A., Engelberg-Kulka, H., & Moll, I. (2011). Selective Translation of Leaderless mRNAs by Specialized Ribosomes Generated by MazF in Escherichia coli Cell, 147 (1), 147-157 DOI: 10.1016/j.cell.2011.07.047

This paper has characterised an interesting new mechanism of stress adaptation in bacteria in which ribosomes are modified to selectively translate a subset of mRNAs that have also been modified by the same enzyme.

Toxin-antitoxin (TA) modules are widespread prokaryotic genetic elements that have generally been characterised as selfish DNA when encoded on plasmids. Chromosomally located TA systems functions are more likely to be integrated into the host cells regulatory networks. mazEF is a well characterised chromosomal TA system in E.coli. The two genes are cotranscribed as an operon; mazE encoding a relatively labile antitoxin that inactivates the more stable endoribonuclease MazF. Under conditions of cell stress mazEF expression is inhibited. As MazE is less stable and degraded by a protease, MazF activity is released. MazF cleaves single stranded mRNAs at ACA sequences hence inhibiting protein synthesis. However this inhibition is not global: about 10% of protein’s synthesis are specifically enabled by MazF action. Some of these protein’s actions are responsible for programmed cell death, others have been shown to permit the survival of a subpopulation of bacterial cells (Amitai et al.2009). This new paper has uncovered the mechanism by which the selective synthesis of this subset of the cell’s proteins is activated by MazF.

Analysing transcripts encoding proteins known to be synthesised in the presence of MazF activity the authors found that they were cleaved at ACA sequences at or closely upstream of their AUG translation start sites. This creates a population of leaderless mRNAs (lmRNAs) that the paper also shows are selectively translated in the presence of MazF activity. Postulating that the selective translation of lmRNAs could  be mediated by MazF modifications to the ribosome itself, the investigators went on to show that MazF also cleaves the 16S rRNA of the 30S ribosomal subunit. This cleavage results in the loss of 43nt from the 3′ end of the 16S rRNA including the anti-Shine Dalgarno sequence (aSD). SD – aSD interactions are important for the initiation of translation of canonical mRNAs with structured 5′ UTRs. However, in this case MazF generates specialised “Stress Ribosomes” lacking the aSD that selectively translate a “leaderless mRNA regulon” also generated by cleavage by MazF.

This paper is important and interesting in that it has discovered an elegant and novel molecular mechanism employed by bacteria during times of environmental stress. It also adds greatly to understanding bacterial programmed cell death and the functions of chromosomally located TA systems both of which are contentious subjects in relation to how their evolution typifies aspects of both altruism and selfishness.