TIBS 25 – AUGUST 2000

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JOURNAL CLUBhypothetical amplification event wouldhelp explain how it is that a small amountof dsRNA is able to destroy a much largerquantity of mRNA, and perhaps also howthe effect can propagate throughout anorganism. The RNA-dependent RNApolymerase might replicate the parentaldsRNA inducer of silencing, but, morelikely, acts on the 21–25-ribonucleotide-long fragments that are associated withsilencing. These short RNAs are known todirect the cleavage of mRNA athomologous sequences, producingfurther RNAs of the same length.

The mechanistic similarity betweenRNAi, PTGS and quelling points to aconserved mechanism evolved by acommon ancestor of animals, plants andfungi. If so, this phenomenon is likely tohave an important physiological role. It iswell known that PTGS is able to protectplants from infection by certain viruses,and it could be that other organisms alsoavail themselves of this anti-viraladaptation. Biotechnologists are alsotaking advantage of the phenomenon as areverse genetics tool, by using silencing toknock out specific genes. Forthcoming

advances in this field, whether in basic orapplied research, will certainly besomething to shout about!

1 Mourrain, P. et al. (2000) Arabidopsis SGS2 and SGS3genes are required for posttranscriptional gene silencingand natural virus resistance. Cell 101, 533–542

2 Dalmay, T. et al. (2000) An RNA-dependent RNApolymerase gene in Arabidopsis is required forposttranscriptional gene silencing mediated by atransgene but not by a virus. Cell 101, 543–553

YEN CHOO

Email: yen.choo@gendaq.com

Snatched from thejaws of life

The central dogma has finally revealedsome of its secrets to the eagerly waitingscientific community. We have been giventhe first high-resolution glimpse of RNApolymerase II (Pol II), the machine thattranscribes the genetic code intomessenger RNA for translation into theprotein polypeptides, which are theworkhorses of the cell. Over the past 20years, Kornberg and colleagues haveapplied almost every known biophysicaltechnique to unravel the details of thisintricate machine that can unwind DNA,translate and proofread, all in one. Thismammoth task has culminated in theexquisitely detailed 3.5 Å structure of tenof the 12 constituent subunits of Pol IIfrom Saccharomyces cerevisiae1.

The architecture reveals a large clamp-like molecule, with the smaller subunits(Rpb3, 5, 6 and 8–12) packed around themassive core comprising Rpb1 and 2,which together account for 70% of thevolume of the molecular envelope. A 20-bp

DNA duplex has been modelled into thedeep cleft between the two core subunits,based on the structure previouslydetermined by 2D crystallography of anactively transcribing complex. At the openend of this cleft, a pair of jaws, composedof an extended region of Rpb1 andsubunits units 5 and 9, clamp around theDNA. Deeper inside the cleft, close by theMn21 active site, Rpb6 and other domainsof subunits Rpb1 and Rpb2 form a mobilearm that is believed to clamp down ontothe transcribed DNA–RNA hybrid and isprobably responsible for the highprocessivity of Pol II. However, at the heartof the machine, Rpb2 blocks the path ofthe unwound transcription bubble, forcingit to divert down a channel to the Mn21

active site. Two pores at the apex of afunnel beneath the active site are thoughtto be the sites of entry of the substrateand elongation factors, which reactivatestalled complexes after backtracking andproofreading. Comparison with therecently solved structures of prokaryoticRNA polymerases shows a remarkabledegree of conservation between the twokingdoms. The conserved regions tend tobe those involved in the DNA–RNA hybrid

interaction, whereas the divergentsubunits are responsible for specializedeukaryotic functions.

Although this could be considered thepinnacle of an odyssey in structuralbiology, the macromolecular crystalstructure is not the end but, paradoxically,the beginning of further challenges tounderstand transcription. This firstglimpse of a eukaryotic RNA polymerasein such detail will provide insight not onlyinto the structure of the related Pol I andPol III molecules but also into the humanenzyme, with which it shares 53%sequence identity. The foundation forunderstanding the complicated regulatorymechanisms of eukaryotic transcription isnow established. Many tiers of otherinteractions need to be assembled beforethe details of the mechanisms, whichunderpin our very existence, are revealed.

1 Cramer, P. et al. (2000) Architecture of RNA polymeraseII and implications for the transcription mechanism.Science 288, 640–649

KATHRYN PHILLIPS

Email: kathryn@cryst.bioc.cam.ac.uk

Journal Club, a new section in TiBS!

This section is designed to provide comment on recentsignificant papers appearing in the primary literature

allowing you to keep up-to-date with TiBS. In addition,many of the contributions have appeared in the News

and Comments section of BioMedNet (access viahttp://bmn.com).