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On Retrons

We reprint this ar­ti­cle from Habib Maroon's blog Biob­a­bel, with his kind per­mis­sion.

by Habib Ma­roon

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, (i.e., 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.

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 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.

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.

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

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 hints of nor­mal func­tion.

Stud­ies on retrons from Vib­rio cholerae sug­gest po­ten­tially im­por­tant roles for ms­D­NAs. Epi­demic cholera is caused by V. cholerae serotypes O1 and O139, both of which con­tain the retron Vc95. Non-O1, non-O139 strains rarely con­tain Vc95. This retron is not as­so­ci­ated with the CTX­phi prophage that en­codes cholera toxin, how­ever it's pres­ence cor­re­lates with path­o­genic­ity. In­ter­est­ingly, the ge­nomic lo­ca­tion of Vc95 is oc­cu­pied by other species of retron or by in­ser­tions of non-cod­ing se­quences in other V. cholerae strains. This im­plies that retrons are in­deed mo­bile el­e­ments, how­ever analy­sis of this site has not yielded many clues about po­ten­tial mech­a­nisms of in­te­gra­tion or ex­ci­sion.

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.

 

Ref­er­ences

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

In­ouye, K., Tan­i­moto, S., Kami­moto, M., Shi­mamoto, T., & Shi­mamoto, T. (2011) Two novel retron el­e­ments are re­placed with retron-Vc95 in Vib­rio cholerae. Mi­cro­bi­ol­ogy and Im­munol­ogy, 55(7), 510–513. DOI: 10.1111/j.1348–0421.2011.00342.x

 

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Nick Heng
14 years ago

This is my first visit to Small Things Con­sid­ered and I find an ar­ti­cle on retrons. It brings back good mem­o­ries be­cause I wrote an elec­tive es­say about msDNA/retrons in 1991 (as a third-year un­der­grad­u­ate stu­dent) due to its highly un­usual struc­ture, and pos­si­bly the first in­stance of retro­trans­po­si­tion in prokary­otes. Back then, its role(s) was un­known and, 20+ years down the track, is still pretty much the same ex­cept for the fas­ci­nat­ing link be­tween the retron and cholera patho­gen­e­sis. Thank you for the ar­ti­cle and for main­tain­ing such a great blog­ging site!

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