Where Art Thou, O Ri­bo­some?

by Elio

In the re­cent post The Age of Imag­ing, I mused about ad­vances in mi­croscopy that are rev­o­lu­tion­iz­ing our con­cept of bac­te­r­ial cell struc­ture. High on the list of amaz­ing de­vel­op­ments is cry­o­elec­tron to­mog­ra­phy (CET), a tech­nique that al­lows one to look in­side bac­te­r­ial cells. Flash-freez­ing cells at very low tem­per­a­tures em­beds them in vit­re­ous ice, and this pre­serves their struc­tures when sub­se­quently sub­jected to the high vac­uum of the elec­tron mi­cro­scope. This process is min­i­mally in­va­sive and re­quires no stain­ing. Bac­te­ria frozen in this man­ner can swim away af­ter thaw­ing! A great tu­to­r­ial about this tech­nique and its uses is pro­vided on Grant Jensen's web page.

Cry­o­elec­tron to­mog­ra­phy of a vit­ri­fied cell show­ing ri­bo­somes and the mem­brane (grey) in an op­ti­cal slice. Ribo­somes de­ter­mined with high fi­delity are green, those with lesser fi­delity are yel­low. Source

Cry­o­elec­tron to­mog­ra­phy has its lim­i­ta­tions, some of which are dis­cussed by Jensen. Most per­ti­nent to bac­te­r­ial mi­croscopy is the thick­ness of the ob­ject viewed. It turns out that bac­te­ria like E. coli are close to the up­per limit of what can be con­veniently looked at, at least for now. Most of the ar­ti­cles writ­ten so far con­cern small­ish bac­te­ria, such as Bdellovib­rio, Caulobac­ter, Flavobac­terium, Spiro­plasma, and Prochloro­coc­cus, among oth­ers. How­ever, stud­ies on sub­cel­lu­lar struc­tures near the cell sur­face or pro­trud­ing from it have been re­ported in larger cells as well. An overview of such stud­ies can be found in a re­cent re­view. One can rea­son­ably ex­pect an avalanche of re­ports of this sort. It's al­ready hap­pen­ing.

How to choose one ex­am­ple from such a rich of­fer­ing? I opted for one that deals with a ques­tion that I wor­ried about long ago: where are ri­bo­somes lo­cated in bac­te­r­ial cells? Are they mostly at­tached to nascent mes­sen­ger RNA strands, mak­ing pro­teins even as the mRNA is be­ing tran­scribed? This ques­tion was re­cently ap­proached by CET us­ing the slen­der my­coplasma, Spiro­plasma mel­liferum. Al­though the full an­swer to my ques­tion is not yet avail­able by this tech­nique, the au­thors showed that most ri­bo­somes seem to be more or less ran­domly dis­trib­uted in the cell. About 40% may well be in polyrib­somes – those strings-of-pearls made of ri­bo­somes at­tached to mRNA. (This fig­ure is lower than that seen in fast grow­ing bac­te­ria, where it is about 80%.) About 15% of the ri­bo­somes ap­pear to be ly­ing close to the mem­brane, with their 50S sub­units fac­ing it. This is the ori­en­ta­tion one would ex­pect if the pro­teins be­ing made are in the process of of be­ing in­serted in the mem­brane. (For a re­port imag­ing this process, click here.) This process, which links tran­scrip­tion, trans­la­tion, and in­ser­tion of pro­teins into the mem­brane, was termed 'transer­tion' by Woldringh.

One more thing. When it comes to ri­bo­somes, their con­cen­tra­tion is highly de­pen­dent on the growth rate as im­posed by the nu­tri­tional con­di­tions. There­fore, pa­ra­me­ters such as the pro­portion en­gaged in trans­la­tion or transer­tion should be stud­ied in cells grow­ing at dif­fer­ent rates. Some of us have been say­ing this for a long time.

 

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