120 TIBS 13-April 1988

extend through four additional unlabeled bases to bring the collective size of the mapped fragments up to that observed for the intact protected region.

To identify binding proteins, the labeled complexes were UV crosslinked and subsequently analysed by SDS gel electrophoresis. A single complex of 100 kDa was obtained. Mowing for bound RNA, this would correspond to a protein of about 85 kDa. Proteinase K treatment prior to UV crosslinking prevented com- plex formation, as did excess unlabeled 5' L-mRNA. Excess unlabeled, non- specific, competitor RNA did not affect the complex. Iron administration to rats did not change the amount of crosslinked complex formed with tissue extracts. Upon iron addition to cultured cells, however, the crosslinked complex increased in parallel with B1. This suggests that the crosslinked protein

may be a component of B1. In B2, the spatial orientation of the binding pro- tein(s) to the RNA may not favor cross- linking.

This work, then, provides the first indication in vitro that iron-sensitive cytoplasmic protein(s) may bind ferritin mRNAs at a specific site and thus regu- late their translation. The most likely target is a 28-base sequence highly con- served among all known ferritin mRNAs. In the rat L-mRNA, it is located 142-169 nucleotides before the AUG start of the polypeptide subunit. This region can be drawn in a stem and loop structure as has been suggested for mRNAs of some translationally- controlled prokaryotic genes (see Fig. 1). At present, however, there is no experi- mental evidence that such a structure exists. Future work on this system should elucidate the mechanism of iron-

mediated translational control of ferritin synthesis and a role, if any, for site speci- fic mRNA-protein complexes.

References 1 Nomura, M., Gourse, R. and Baughman, G.

(1984) Annu. Rev. Biochem. 53, 75-117 2 Theft, E. C. (1987)Annu. Rev. Biochem. 56,

289-315 3 Aziz, N. and Munro, H. N. (1987) Proc. Natl

Acad. Sci. USA 84, 8478-8482 4 Hentze, M. W., Caughman, S. W., Rouault,

T. A., Barriocanal, J. G., Dancis, A., Harford, J. B. and Klausner, R. D. (1987) Science 238, 1570-1573

5 Leibold, E. A. and Munro, H. N. (1988) Proc. Natl Acad. Sci. USA 85, 2171-2175

6 Leibold, E. A. and Munro, H. N. (1987) J. Biol. Chem. 262, 7335-7341

7 Murray, M. T., White, K. and Munro, H. N. (1987) Proc. Natl Acad. Sci. USA 84, 7438- 7442

Features 'Omnipotent' nonsense

suppressors: new clues to an old puzzle

Ancirei P. Surguchov

Genetic characterization of omnipotent nonsense-suppressors sup/and sup2 in yeast originally suggested that they code for termination factors, but their role in the control of translational fidelity and their effect on other ribosomal properties seemed to indicate that their products are ribosomal proteins. However, recent sequence data dem- onstrated that one of them (sup2) is highly homologous to an elongation factor gene. What is the function of the polypeptide products coded for by these genes and the

reasons for these contradictions?

Persistently puzzling suppressors Recessive suppressors of nonsense co-

dons in yeast Saccharomyces cerevisiae were first described about 25 years agoL These suppressors, designated supl and sup21.2, or sup35 and sup453 , were found to be effective towards all three types of nonsense codons (UAA, UAG and U G A ) 4'5. They are therefore called 'om- nipotent' suppressors, supl is located on chromosome II, whereas sup2 is on chromosome IV (Ref. 5). Both genes are essential for cell growth and inactivation of either of them by mutation or gene

A. P. Surguchov is at the Institute of Experimental Cardiology, USSR Cardiology Research Center, 3rd Cherepkovskaya Street 15,4, Moscow 121552, USSR.

~) I088, Elsevier Publications Cambridge 0376- 5067/88/$02.00

disruption is lethal. The properties of recessive suppressors s dearly suggest that both supl and sup2 genes do not code for tRNA, as dominant suppressors do, but rather for proteins participating in the process of translation. Genetic characteristics of the recessive suppres- sors (e.g. the spectrum of nonsense sup- pression, interaction with dominant, and tRNA-mediated suppressors) led to the proposal that they may code for termina- tion factors of protein synthesis or pro- teins responsible for termination factors binding to the ribosomes 2.

The first clues as to how these suppres- sors work were discovered when analysis of cell-free translation systems prepared from these strains showed elevated mis- reading of mRNA compared with the

parent strains~. However, attempts to identify the suppressor gene products gave conflicting results. On the one hand, several properties of ribosomes in the strains carrying recessive suppressor mutations were found to be changed: • the level of ribosomal ambiguity is increased4-~; • the interaction of ribosomes with anti- biotics is altered, e.g. association con- stant K a for cydoheximide is increasedS; • the level of ribosomal subunits in cell- free extracts is elevated4,5; • biogenesis of ribosomal subunits is affected s.

These data seemed to point to ribo- somal proteins as the polypeptide prod- ucts of the suppressor genes. However, all attempts to find a mutant ~bosomal protein with an altered electrophoretic mobility by two-dimensional elec- trophoretic techniques were unsuccess- fuP except for one report concerning the sup35 (supl) gene 6. Moreover, some properties of the suppressor genes were inconsistent with the conclusion that their products are canonical ribosomal proteins: • the size of the suppressor genes coding region is longer7-9 than all known yeast ribosomal protein genes; • the rate of transcription for supl at least is significantly lower than for ribosomal protein genesg; • the codon usage for the suppressor genes differs from that of ribosomal pro- tein genes7.10.

TIBS 13-April 1988 121

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Fig. 1. Comparison of the amino acid sequences of the sup2 polypeptide product, yeast EF-Irt , mtEF-Tu and E. coil. EF-TuA. Fragments of the polypeptides containing GTP- and aminoacyl-tRNA-binding conservative domains are aligned to give maximal homology by introducing several gaps (-) . The regions of exact homology and conservative substitutions between the sup2 product and either elongation factor are indicated by boxes. Conservative domains G I--G5 involved in GTP-binding are indicated by solid lines, whereas region T (important for aminoacyl-tRNA binding) is shown by a dashed line. Positions, where there is homology between EFs but not to the sup2 product are marked by an asterisk (*). For further details see Ref. 10.

Thus, the first puzzle of omnipotent suppressors was that they appear to com- bine the properties of ribosomal proteins and soluble translational factors. Clon- ingg, 11 and sequencingT,10 of the sup- pressor genes provided a clue to the understanding of their structural fea- tures and a possible reason for the con- tradictions mentioned above.

Primary structures of the suppressor genes and their products

The nucleotide sequence of sup2 gene 10 revealed a high homology to the family of elongation factor genes from different sources: yeast cytoplasm12, yeast mitochondria 13, Escherichia coil 14 and other organisms. The sup2 gene product is 62% homologous with yeast EF-la and 43% with yeast mitochondrial EF-Tu (for details see Ref. 10). How- ever, sup2 is not szmcturally identical to the EF-la gene coding for the yeast cytoplasmic elongation factor ]°,12 and isolation of conditional lethal mutants (see below) for the sup2 gene 5 confirmed that these two genes are not functionally interchangeable. It is noteworthy that earlier nonsense-suppressor activity was shown for mutant forms of bacterial elongation factor EF-Tu zS. The highest homology between the sup2 gene prod- uct deduced from the nucleotide sequence and elongation factors was found in GTP- and aminoacyl-tRNA- binding domains 1° indicating that these regions are highly conserved (Fig. 1). This is presumably due to the evolution- ary constraints for the part of the molecules involved in the recognition of these ligands. Thus, the sup2 gene prod- uct may be related to GTP-binding (G) proteins. Attention has recently been focused on these proteins due to their critical involvement in vital cell processes such as signal transduction; new proteins

belonging to the family are being discov- ered continually. In spite of rather high homology of the sup2 gene product to EF-la the suppressor gene does not appear to be highly expressed since the pattern of codon usage is more similar to yeast genes expressed at a low level than to genes encoding abundant proteins such as ribosomal proteins or transla- tional factors required in each cycle of elongation. The sup l gene product was also found to have homology to proteins involved in nucleotide- and tRNA-bind- ing (aminoacyl-tRNA synthetases, ras proteins) 7.

Possible tlumetians ofthe suppressor genes If we combine the data on the partici-

pation of omnipotent suppressor genes in the control of translational fidelity ~6 with the localization of the nucleotide- binding and aminoacyl-tRNA- (or tRNA-) binding domains 7,l° in their polypeptide products, it is tempting to suggest that their function (or one of their functions) is a nucleotide-depen- dent proofreading process ofcodon-anti- codon interactions. A similar function has been suggested previously for bac- terial elongation factor EF-Tu 16.

The identification of the suppressor gene product as a member of the elon- gation factor family in yeast points to the lack of our knowledge of the detailed mechanism of translation in this organism. Although yeasts are fre- quently used as a convenient model organism for the study of many biologi- cal processes, the mechanism of protein synthesis, and particularly the involve- ment of the soluble protein factors in this process, are characterized very super- ficially in yeast compared to bacterial or mammalian systems. In addition, the results concerning omnipotent suppres- sor genes suggest that certain aspects of

eukaryotic translation are different from prokaryotic systems. Proteins coded by suppressor genes apparently combine the characteristics of termination factors, ribosomal proteins and elongation fac- tors (recognition of nonsense codons, control of ribosomal ambiguity, struc- tural similarity to elongation factors) making it difficult to assign them to just one of these classes of proteins. The lack of exact criteria and definitions of these three protein classes in yeast compounds this problem.

In the course of the primary charac- terization of omnipotent suppressor mutations several other genes have been describedS. 17-19 that affect the efficiency of suppression by supl and sup2. These include antisuppressors which lower, and allosuppressors which increase the efficiency of the omnipotent sup- pressors. The identification of the prod- ucts of these modifier genes as new trans- lational factors or ribosomal proteins, interacting with suppressor gene prod- ucts or another class of proteins is the challenge for the future.

How did suppressor genes arise? Although supl and sup2 appear to

have similar functions they have dissimi- lar primary structures. Until the results of sequencing became available, all the data seemed to indicate that the two genes were similar. The suppressor mc~tations in these two genes are reces- sive, the genes possess common func- tions being omnipotent suppressors of nonsense mutations, their pleiotropic effects (e.g. high or low temperature sen- sitivity, high osmosensitivity, respiratory deficiency) are very similar 4,5. All these data seemed to indicate that they had evolved from a common ancestor gene and may possess structural homology.

However, a comparison of the cloned

122

Table L Comparison of supl and sup2 genes 7-t°

supl sup2

Nudeotide sequences typical forintrons Length of the transcripts Repeats of nucleotide sequences Homology to elongation factor family

absent present 1.5-1.6kb 2.3-2.5 and 1.4kb absent located in 5' end of coding region absent present

supl and sup2 genes sequences and deduced polypeptide products revealed strikingly different features of their pri- mary structures and organization 7-1° (Table I). The analysis of structural fea- tures of the genes suggests that they did not evolve from a common ancestor gene. Moreover, we believe that their ancestoral genes were located in differ- ent intracellular genomes, i.e. those of the nucleus and mitochondrion. We propose that the supl gene evolved from a mitochondrial predecessor, since • it possesses rather high A + T content (828 A + T compared to 486 G+C); • its codon usage is similar to that of mitochondrial genes (see Table II); • computer-assisted comparison of its structure revealed a homology with sev- eral mitochondrial genes 23.

The supl gene might have been trans- ported from the mitochondrial to nuclear genome by a process described previ- ously 24-26. In contrast, sup2 and EF- la presumably evolved from a common nuclear ancestor after its duplication. A similarity of the functional properties of supl and sup2 genes may be explained by the evolutionary adaptation of their products to the interaction with the same sets of molecules: tRNAs, nucleotide(s), ribosomal components.

Why are there two genes? Intriguing questions are: why two

genes (supl and sup2) which possess

apparently similar functions and mu- tational patterns are present in the yeast genome, and what equivalent genes exist in other eukaryotes? The answers to these questions still present a puzzle. A great number of conditional lethal mu- tants for both sup1 and sup2 genes have been obtained 5. The majority of these mutants possessed high-temperature sensitive (Hts-) phenotype, being unable to grow at elevated (36°C) tem- perature. Hts- suppressor strains were isolated after checking the ability of many omnipotent suppressors to grow at 36°C. Since conditional lethal mutants were obtained for both genes, they are dearly both required for cell survival and are not fully identical functionally. Indeed, duo cum faciunt idem, non est idem.

There are indications 5 that supl and sup2 genes encode not only cytoplasmic, but also mitochondrial protein products. Many omnipotent suppressor strains were found to be respiratory deficient. A spectrophotometric analysis of the cyto- chrome spectra as well as the discovery of a differential chloramphenicol effect on mitochondrial protein synthesis in suppressor and parent strains demon- strated that mutations in sup1 or sup2 affect protein synthesis both in cyto- plasm and mitochondria 5,27. Therefore, supl and sup2 may belong to the family of nuclear genes that code both cytoplas- mic and mitochondrial proteins with

Table 11. Preferred codons in yeast mitochondrial proteins and protein components of translational apparatus

Amino Preferred codon acid

sup l a Mitochondriai proteins b

Asn AAT AAT Tyr TAT TAT Leu T T A T T A His C AT C AT Asp GAT GAT

Yeast elongation Yeast cytoplasmic Tyrosyl- tRNA factor EF- lac ribosomal synthetase e

proteins d

AAC AAC AAC TAC TAC TAC TTG TTG TTG CAC CAC CAC GAC GAC GAC

a Ref. 7. bData summarized for ATPase subunits 6 and 9, cytochrome oxidase subunits 1, 2 and 3 and

als3cytochrome b 20. c Ref. 12. d Ref. 21. eRef. 22.

TIBS 1 3 - A p r i l 1988

similar functional properties, that is they may be genes with double coding capac- ity 2s-3o. If this is so, further study of the structural and functional properties of these genes will provide an exciting opportunity to elucidate the detailed mechanism of protein synthesis in both the cytoplasm and mitochondria, and the coordination of this process in these two cellular compartments. Thus, there are reasons to believe that further study of omnipotent suppressors will provide insights into etiology of the previously described contradictory findings.

References 1 lnge-Vechtomov, S. G. (1964) Vestnik LGU,

Set. Biol. (USSR) 2,112-115 2 Inge-Vechtomov, S. G. and Andrianova,

V. M. (1970) Genetika (USSR) 6,103-115 3 Hawthorne, D. C. and Leupold, V. (1974)

Curt. Top. Micobiol. lmmut~oi. 64,1-47 4 Surgchov, A. P., Berestetskaya, Yu.V.,

Fominykch, E. S., Pospelova, E. M., Smirnov, V. N., Ter-Avanesyan, M. D. and Inge-Vech- tomov, S. G. (1980) FEBS Lett. 111,175-178

5 Surguchov, A. P., Smirnov, V. N., Ter-Avan- esyan, M. D. and Inge-Vechtomov, S. G. (1984) in Physicochemical Biology Reviews Vol. 4 (Skulachev, V. P., ed.), pp. 147-205, Har0vood Academic Publishers

6 Eustice, D. C., Wakem, L. P., Wilhelm, J. M. and Sherman, F. (1986) I. Mol. Biol. 188,207- 214

7 Breining, P. and Piepersberg, W. (1986) Nu- cleic Acids Res. 14, 5187-5197

8 Surguchov, A. P., Telkov, M. V. and Smirnov~ V. N. (1986) FEBS Lett. 206, 147-150

9 Himmelfa~b, H. J., Maicas, E. and Friesen, J. D. {1985) Mol. Cell. Biol. 5,816-822

10 Kushnirov, V. V., Ter-Avanesyan, M. D., Sur- guchov, A. P., Smirnov, V. N. and lnge-Vech- tomov, S. G. (1987) FEBS Lett. 215,257-260

11 Breining, P., Surguchov, A. P. and Piepersberg, W. (1984) Curt. Genet. 8, 467--470

12 Nagata, S., Nageshima, K., Tsunetsugu- Yokota, Y., Fujimara, K., Miyazaki, M. and Kaziro, Y. (1984) EMBO J. 3,1825-1830

13 Nagata, S., Tsunetsugu-Yokota, Y., Naito, A. and Kaziro, J. (1983) Proc. Natl. Acad. Sci. USA 80, 6192-6196

14 Yokota, T., Sugisaki, H., Takanami, M. and Kasiro, Y. (1980) Gene 12, 25-31

15 Vijgenboom, E., Vink, T., Kraal, B. and Bosch, I.. (1985)EMBOJ. 4,1049-1052

16 Hopfield, J. J. (197,~) Proc. Natl Acad. Sci. USA 71, 4135--4139

17 Liebman, S. W. and Cavenagh, M. M. (1981) Curr. Genet. 3, 27-29

18 Liebman, S. W., Cavenagh, M. M. and Ben- net, L. N. (1980)./. Bacteriol. 143,1527-1529

19 Liebman, S. W. and Cavenagh, M. M. (1980) Genetics 95, 49--61

20 Bonitz, S. G. and Corrusi, G. (1980) J. Biol. Chem. 255,11927-11941

21 Leer, R. J., van Raamsdonk-Duin, M. M. C., Nagendoorn, J. M., Mager, W. N. and Planta, R. J. (1984) Nucleic. Acids Res. 12, 6685--6700

22 Winter, G., Koch, G. L. E., Hartley, B. S. and Barker, D. (1983) Fur. J. Biochem. 132, 383- 387

23 Surguchov, A. P. Life Science Advances (in press)

TIBS 13 -April 1988 123

24 Farrely, F. and Butow, R. A. (1983) Nature 301, 2'~-301

25 Boogamt, P., SamaUo, J. and Agsteribbe, E. (1982) Nature 298,187-189

26 Jambs, H. T., Posakony, J. W., Grula, J. W., Roberts, J. W., Xin, J. H., Britten, R. J. and

Davidson, E. H. (1983)./. Mol. Biol. 165,609- 632

27 Surguchov, A. P., Sudarickov, A. B., Telckov, M. V., Smirnov, V. N., Ter-Avanesyan, M. D. and Inge-Vechtomov, S. G. (1983) Mol. Gen. Genet. 189,172-174

28 Surguchov, A. P. (1987) Trends Biochem. Sci. 12, 335-338

29 Natsoulis, G., Hilger, F. and Fink, G. R. (1986) Cell 46, 235-243

30 Wu, M. and Tzagoloff, A. (1987) J. Biol. Chem. 262,12275-12282

Emerging Techniques

A new non-crystallographic imafle-processinfl technique

reveals the architecture of fibosornes

Joachim Frank, Adriana Verschoor, Terence Wagenknecht, Michael Radermacher and Jose-Maria Carazo

Due to a recent breakthrough in three4bnmsional reconsmc~n techniques, macro- molecules or macromoleodar assemblies owur~ng in non-crystalline form as 'single ptmides' can now be reconstn¢~ with high statiai~ definition from low-dose elearon micrographs. The new technique has been used tiros far to detemmze the sauaures of the 50S ribosomal subunit and the 70S dbosome from Escherichia coil, as well as the 40S subunit and the 80S ribosome from rabbit retiadocytes. The technique will also enable the mapping of surface ligands through direct three-dimensional vimalization, and can #ms be erpected to make a major contribution to an understanding of the function of ribosomes, arm potentially many

other important macromolecular assemblies.

Electron microscopy at the macro- molecular level encounters many ob- stacles, a serious one being the damage inflicted on the specimen by the electron beam. Low-dose techniques have been devised to deal with this problem, but the resulting statistical definition of the micrographs is so poor that images of a large number of molecules occurring in the same orientation on the specimen support must be averaged. This is readily achieved when the molecules occur ordered in a thin crystalline sheet. Images of such a crystal can be averaged by Fourier analysis, and many such aver- ages obtained with the crystal tilted over a wide range of angles can be combined into a three-dimensional reconstruc- tion I. Unfortunately, many types of macromolecular assemblies cannot be brought into such a highly ordered form, and thus are notamenable to this type of analysis.

Z Frank, A. Verschoor, T. Wagenknecht, M. Rader- reacher and J-M. Carazo are at the Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany, NY 12201, USA.

Imaging and two-dimensional image processing of single molecules

For some time we have been exploring methods of computer image analysis that allow images of single nlacromolecules to be averaged 2-4. Initially, this approach was applicable only to particles that show a preferred orientation on the spechven support. For averaging to be done, images of molecules showing the same face must be placed in equivalent posi- tions in their reference frames; this is achieved through the use of translational and rotational cross-correlation align- ment techniques. Essentially, two images to be aligned are shifted and rotated rela- tive to each other in the computer, and in each position the cross-correlation coefficient of all superimposed elements is computed. Optimum alignment (Fig. la) is indicated by a maximum of the cross-correlation.

Implementation of this averaging approach is obviously limited, since it further requires a population of molecules that is quite homogeneous in terms of particle structure if the average is to be meaningful. In practice, molecule projections observed in the

electron microscopic experiment gener- ally show a host of variations, which may be due to preparation artifacts, radiation damage, differing orientations, or physiologically relevant conformational differences. Therefore, the averaging must be preceded by a decision as to which particles are to be included and which to be rejected, or the more com- plicated decision of whether the particles fall into subgroups that should be aver- aged separately.

Initially this decision was made by visual inspection of the images. The introduction of multivariate statistical analysis into this field by Van Heel and Frank5 presented a breakthrough, since it provided the means by which molecule images could be grouped according to objective criteria. The variations among a given set of images are mapped on to independent components of variation, called factors, which are ordered by importance. As a result, each image can be represented by a point, or a set of coordinates in a space (factor space) that is spanned by a few most significant fac- tors. The grouping of images in that space is then assessed by the use of class- ification techniques. In the case that most interests us here, the observable variations are due to the fact that the par- ticles lie in different orientations on the specimen grid. Images grouped in the same part of factor space then represent particles having very similar orientations (Fig. lb).

In sunm;~y, the correlation align- ment techniques combined with multi- variate statistical :~nalysis and classifica- tion techniques a~ow a homogeneous set of particle images with defined orienta- tions to be selected from micrographs.

The single-exposure random-conical tilt reconstruction method

Despite this very significant progress in the analysis of particle projections, a practical and convincing method of three-dimensional reconstruction of single macromolecules did not emerge until recently. For such a method to be useful, it should allow low-dose imaging so as to minimize radiation damage to

~) 1988. Elsevier Pubfications Cambridge 0376- 5067/88/$02.00