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References 1 de Duve, C. (1969) Proc. Roy. Soc. Set. B. 173, 71-83 2 Beevers, H. (1969) Ann. N.Y. Acad. Sci. 168, 313--324 3 Hruban, Z. and l~echeigl, M. (1969) Int. Rev. Cytol. (Suppl. 1) 1-296 4 Palade, G. E. (1975) Science 189, 347-358 5 Chua, N. H. and Schmidt, G. W. (1979) J. Cell. BioL 81,461-483 6 Schatz, G. (1979) FEBS Len. 103, 203-211 7 Lodish, H. F. and Rothman, J. E. (1979) Sci. Am. 240, 38-53 8 Hock, B. (1974) P/anta 115, 271-280

9 Bowden, L. and Lord, J. M. (1976) Biochem. J. 154, 491-499 10 Mellor, R. B., Krusius, T. and Lord, J. M. (1980) Plant Physiol. 65, 1073--1075 11 Hanover, J. A. and Lennarz, W. J. (1980) J. Biol. Chem. 255, 3600--3604 12 Beevers, H. (1979)Ann. Rev. Plant Physiol. 30, 159-193 13 Wirtz, K. W. A. (1974) Biochim. Biophys. Acta 344, 95-117 14 Redman, C. B., Grab, D. J. and Irukulla, R. (1972) Arch. Biochem. Biophys. 152, 496501 15 Lazarow, P. B. and de Duve, C. (1973) J. Cell Biol. 59, 507-524

promoter site within the abundance of unspecific sites, and what are the pertinent structural features of the two macromolecules that interact in this process?

The interaction of E. coli RNA polymerase with promoters Hermann

Bujard

Among protein-nucleic acid interactions promoter recognition by RNA polymerases is o f special interest since it represents an important step in the controlled flux of genetic information common to all biological systems. During gene expression information is transferred from its storage site - usually a double-stranded DNA - to its site of action, usually a protein. Since only a fraction of the vast biochemical potential of a cell is used at any given time this flux of information must be controlled at various levels. An important level for such control mechanisms is transcription, which can be divided into: (a) the enzymatic processes by which a predetermined sequence of monomeric units is transferred with high fidelity from the DNA template to the RNA that will be used as a template for protein synthesis; (b) the recognitory processes by which the transcriptional machinery recognizes start and stop signals encoded in the DNA template. Selectivity of transcription is due to the recognitory events flanking actual R N A synthesis• In the simplest case a transcriptional unit consists of a start signal - a promoter - the transcribed region, which may comprise the information for one or more polypeptide chains and a stop signalthe terminator (Fig. 1). The extent to which the transcriptional unit is expressed depends primarily upon the efficiency with which the promoter is utilized by the DNA-dependent RNA polymerase. In E. coli we have examples of such simple control mechanisms (for instance the I gene of the lac operon producing constant levels of Hermann Bujard is at the Molekulare Genetik, Universitlit Heidelberg, Im Neuenheimer Feld 230, D-69 Heidelberg, F.R.G.

16 Goldman, B. M. and Blobel, G. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 5066--5070 17 Blobel, G. and Dobberstein, B. (1975) J. Cell Biol. 67, 852-862 18 Walk, R. A. and Hock, B. (1978) Biochem. Biophys. Res. Commun. 81,636--643 19 Reizman, H., Weir, E. M., Leaver, C. J., Titus, D. E. and Becker, W. M. (1980) Plant Physiol. 65, 40-46 20 Gonzalez, E. and Beevers, H. (1976) Plant Physiol. 57, 406-409 21 Lord, J. M. and Bowden, L. (1978) Plant Physiol. 61,266-270 22 Mellor, R. B., Roberts, L. M. and Lord, J. M. (1980) J. Exp. Bot. (in press)

repressor molecules). In many cases, however, the signals encoded around the transcriptional unit are targets for additional regulatory elements• Thus, negatively or positively acting elements (e.g. repressor or catabolite gene activator protein (CAP) molecules) can diminish or enhance promoter activity, while termination, and anti-termination, factors can influence the efficiency of transcriptional stop signals. These superimposed regulatory mechanisms permit the cell to respond to environmental changes (Fig. 1). Here I shall discuss primarily the interaction of E. coli R N A polymerase with promoter sites, paying less attention to modulation of this interaction by additional effectors. Before R N A synthesis is initiated, the enzyme must recognize a promoter, form a complex and find the information for the precise positioning of the first nucleotide in the nascent R N A chain. The basic questions about this recognitory process therefore are: how does RNA polymerase find a

The enzyme DNA-dependent RNA polymerase of E. coli has a molecular weight of 500,000• This so-called holoenzyme consists of five subunits /3, /3', or2 and o-. It can be dissociated into the core enzyme,/3,/3', a2, which is able to perform the basic enzymatic processes of RNA synthesis but does not recognize promoters. The o--subunit is important for specific recognition 1, although it does not alone bind to DNA 2. Little is known about the function of the other subunits except for /3 which is intimately involved in the catalytic processes of RNA chain initiation and elongation, since, in connection with the a subunit, it binds rifampicin and streptolydigin, drugs known to inhibit these reactions respectively 2. Furthermore, mutations rendering cells resistant to rifampicin have been mapped within the gene of the/3 protein 2. The enzyme--DNA complex E. coil RNA polymerase binds to a large variety of promoters in vitro, forming a stable complex in which the DNA in the promoter region is unwound about one helical turnL The stability of many of these complexes has facilitated the study not only of promoter structure but also of the

Promoter

Terminator

I A

I

/ [ ",,

Activators

Repressors

RNA-Polymerase

Transcript (m-RNA] B

I

-- I

I

C

!

....

I

TerminationAntitermination Factors

Fig. 1. The transcriptional unit. A stretch o f DNA gwing rise to a contiguous piece o f RNA, the transcript, which may code for one or more polypeptide chains (A, B, C). Such a unit is delimited by a start and a stop signal, promoter and terminator, which are recognized by the DNA-dependent RNA polymerase. Both promoter and terminator are prime targets for additional regulatory factors which may enhance or diminish the interaction between the enzyme and the signal encoded in the D NA.

© Elsevier/North-HollandBiomedicalPress 1980

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interaction between the enzyme and DNA. Thus, in the so-called filter binding tech+20 +1 -20 -40 -6O nique 4 DNA fragments bound to polymer! ase are retained by nitrocellulose whereas Pol ymerose non-complexed double-stranded DNA is -, "Foot pri nt" not. Using the filter binding technique protected --,. t= half-lives of polymerase-DNA comple×es p-DNA regions of between several minutes and many m-RNA ~> hours (at 37°C and intermediate ionic Generalized 3" strength) have been found 5'6. 5" promoter -I0 +I Such stable complexes can be utilized for -43 -35 probing biochemical and chemical properAA ties. Thus, D N A fragments pro~Lected by Simplified 3" R N A polymerase against DNAase I diges5' Ioc-promoter tion were isolated and their nucleotide A sequences determined 7. The size of the "Melted"region so-called 'p-DNA' (protected DNA) is "open" around 42 nucleotides and covers the Regions of promoter region from about +20 to - 2 0 mojor contact (Fig. 2, sites within the nucleotide sequences of a promoter will be :identified G" p henceforth by negative or positive numbers depending on whether their position is upstream or downstream from the initiat- Fig. 2. The major topographic properties o f an E. colipromoter-RNA polymerase complex. The top part o f the ing nucleotide of the transcript). Although figure shows the DNA fragment protected by RNA polymerase against limiting digestion with DNAase 1 ('footthe analysis of p-DNA has led to important print') as well as the region inaccessible even under conditions o f vigorous attack by this enzyme ('p-DNA'). In the middle part o f the figure a simplified version o f a generalized promoter sequence is depicted. By conveninsights about promoter structure some properties remain puzzling: the p-DNA- tion, only one DNA strand - the non-coding strand - is shown. Transcription is therefore initiated from lefi to right at position +1, the starting nucleotide o f the nascent mRNA chain. This generalized promoter scheme emphasizes enzyme complex can initiate R N A syn- the regions where the main sequence homologies are found (boxed-in areas): the so-called "Pribnow box' at -10, thesis in the presence of nucleotide triphos- the - 3 5 region, as well as the CA T sequence at the RNA start, and the A T-rich stretch around position -43. The phates and produce a short 'run off' RNA. latter homology is predominantly found in promoters o f high signal strength (e.g. promoter o f coliphage T5, Ref. When the enzyme is dissociated from its 10). The line below shows a small part o f the lac-promoter sequence to demonstrate the effect o f mutations in highly short template, however, it is not able to conserved regions. The transition in the - 1 0 region from the wild type sequence TA TGTT to the one o f the ps rebind to p-DNA, indicating that regions ( TA TA TT) and UV5 (TA TAA T) matation results in a 1 O- and 25-fold increase oflac expression" respectively as important for complex formation were not the ideal' - 1 0 sequence" is approached (,~ 'promoter-up' mutations). The fact that an exchange of a GC for an A T protected against .DNAase I. By develop- base pair does not necessarily cause an increase in promoter strength, despite the high A T-content o f strong proing the so-called 'foot printing' method moters, is demonstrated by the 'promoter-down' mutation ( ~ ) in the - 3 5 region, where the transition from the Schmitz and Galas 8 have shown that E . c o l i favoured A CA sequence to A A A reduces lacoperon expression. (W. Reznikoff,personalcommunication). The bottom part o f the figure depicts where the double helix is drastically distorted upon binding o f the RNA RNA polymerase actually covers more or polymerase ('open' region) and where the major contacts are made between the enzyme and the DNA. It also shows less tightly a region of 70 to 80 base pairs where the lac promoter and the subunits tr and fl of RNA polymerase can be crosslinked indicating the positions of (bp) from about position +20 to - 6 0 (Fig. the respective subunits within the promoter-enzyme complex. 2). Interestingly, one of the DNA strands is protected less efficiently by the enzyme a hexamer in the - 1 0 region. The pro- moters, known to require additional facthan the other, indicating a unique orienta- totypic sequence is TATAAT_, in which the tors for their function, show little or no tion of the template-bound protein. last position, occupied by a thymine, is homology to the prototypic sequence of the identical in all promoters investigated so - 3 5 region, although this finding cannot Promoter sequences far. Also very stringently conserved are the be generalized. Common features might also be p-DNA fragments have provided the first two positions (TATAAT) in contrast expected in the sequence surrounding the first defined probes for the sequencing of to the remainder of the hexamer, where a site of chain initiation, since this reaction is somewhat larger variation is observed. A promoters, and homologies wil:hin such highly selective with respect to the initiatsecond region of homology has been idensequences were identified first by Schaller ing nucleotide: thus R N A chains start pretified around position -351°'11. Here a et al. 7 and Pribnowg: they pointed out a redominantly with a purine, A occurring highly conserved trinucleotide TTG can be gion of relative homology centred around more often than G. In some instances, the - 10 position, quite frequently referred derived fro'm sequence compilation, prohowever, a C or U is the initiating nuto as a 'Pribnow box' (Fig. 2). About 60 vided that the distance between the Pribcleotide although the template would pronow box and the 3 5 region is allowed to promoters have now been sequenced (for review and compilation of 46 sequences vary by two base pairs (Figs 2 and 3). The vide an A- or G-start in neighbouring posisee Ref. 10) and although we have little TTG sequence is followed downstream by tions. Despite this specificity, the starting information about the signal strength of the three less stringently conserved nu- nucleotide is located within a rather poorly various sequences and their possible cleotides and the resulting hexamer conserved sequence (CAT) 6 t o ' 9 bp dependency upon additional effectors, a T T G A C A is part of a stretch of 12 nu- downstream of the - 1 0 hexamer. The number of common structural features can cleotides in which additional homologies highly selective but variable location of this be found. The most conserved sequence is can be found. Interestingly some pro- starting nucleotide in close proximity to the ,

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T I B S - October 1980

most conserved region of a promoter may appear paradoxical. It indicates, however, a sequence-dependent fine adjustment of the initiation region with respect to the reaction centre of the enzyme (there are a few cases where R N A chains are initiated at multiple sites; in most of them, however, the starting sequence consists of several identical nucleotides like G G G or CCC, (see Ref. 10). Promoter mutation

Picking at random one of the published promoter sequences, the 'unprejudiced' observer might have difficulties in following some of the above conclusions (Fig. 3). Caution is indeed indicated as long as our knowledge of the functional implications of primary structure is as poor as it is today. In the case of E. coli promoters, however, the respective conclusions are not only based on an impressive number of welldocumented sequences, there is also firm genetic evidence: of 34 promoter point -43

mutations investigated, 29 fall within the defined regions at - 1 0 and - 3 5 . In addition 14 of the 16 mutations identified within the - 10 hexamer cluster in the three most conserved positions emphasizing the importance of these sites. The most striking results were obtained by analysing some of the 'promoter-up mutations' which increase the efficiency of the lac promoter (Fig. 2). This promoter has in its wild type form the following - 10 hexamer: T A T G T T . A change of the G/C pair in the fourth position of the hexamer increases the promoter efficiency ten-fold. Since polymerase binding unwinds a promoter about one helical turn and induces a local melting near the - 10 position (see below), a change from GC to A T pairs, which reduces hydrogen-bonding, would be expected to have a positive effect on the signal strength of a promoter sequence. However, in the lac U V 5 promoter mutation the original lac - 10 hexamer is converted into the prototypic sequence

-35

T A T A A T increasing the promoter efficiency by another factor of 2.5. Here a change from TA (in the fifth position of the hexamer) to AT, which is not believed to influence the helix stability, still has a profound effect, indicating the importance of specific sites for nucleotide-protein interaction la. Another interesting 'upmutation' is in the - 3 5 region of the lac I gene promoter, where change of G C G to G T G in the region of the T T G prototypic sequence increases promoter activity tenfold TM. Most of the promoter mutations ('down-mutations') diminish promoter activity and although the effects of the various base pair changes are difficult to interpret in all cases, it is obvious that in some positions there is a clear selection for a particular base and any change has adverse effects. In other positions, however, it appears that there is a strong selection against a particular base, and any of the remaining possibilities do not adversely affect promoter activity. - 10

'T T G C A C G A A C C A T A T G T I A A G T A

+ 1

XPRE

GCCTCGTTGCGTTTGT

araC

GCCGTGA TTATAGAC

ACTTTTGTTACGCGTTTT

TGTCA

~A2

AAACAGGT

ATTGACA

ACATGAAGTAACATGCAG

TAAGA TACAAATC(~3

Str

TGTATAT

TCTTGAC

ACCTTTTCGGCATCGCCC

TAAAA

ec

ACCCCAG GCTTTACA

~P25

AAAAATT

TATTTGCT

~P26

AAAATTT

CAGTTGCT

TTGACA

TTCCTT(~)A GGCTTT~GT

TTCGGC@TCC

C T T T A T G C T T C C G G C T C G T A T G T T G T GT G ( ~ ) A T

TCAGGAAAATTTTTCTG TAATCCTACAATTCTTGA

-

15-18bp.

ATAA TAGATTC~TA TATAA

A T T C T C(~)T A

- TATAAT.,---.5-7bp--~C(~)T

Fig. 3. The nucleotide sequence o f selected promoters. The sequences o f the ara D promoter ~aand the PRE promoters o f phage h ~ are examples where to predict a function from sequence information alone appears impossible. In contrast, significant homologies within the conserved regions (around - 1 0 and -35) are found within the promoters depicted in the middle part: T7 A2 from coliphage T7 ~, Strand lac from E. coli~.2L The two promoters o f coliphage T5 (See Ref 10) exhibit sequences o f high signal strength. They show a high overall A T content, an 'ideal' - 1 0 region (TA TAA T) and a striking A T-rich region around -43. The generalized sequence indicated at the bottom is derived from about 40 promoter sequences.

277

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Contacts between RNA polymerase and promoters The stable association 'between RNA polymerase and promoters in vitro permits studies of the complex with highly specific alkylation reactions: methylation of purines with dimethylsulphate TM and ethylation of the backbone phosphates with ethylnitrosourea 14. Two types of question can be answered with such experiments: (a) Which positions within a promoter sequence are essential for the formation of the complex or its stability? (b) Which are the positions within a promoter-polymerase complex where the protein is close enough to the DNA to interfere with the chemical attack, either diminishing or enhancing alkylation? DNA is therefore either alkylated first and its polymerase-binding properties are recorded, or it is alkylated after formation of the enzyme-DNA complex. A'~[kylation patterns obtained in such experiments carried out mainly with lac and phage T7 promoters ~5-a8 revealed three regions of extensive contact between RNA polymerase and promoters: the 'Pribnow box', the - 3 5 region and the sequence around the RNA initiation sites (Fig. 2). This is in excellent agreement with the picture derived from sequence homologies and analyses of promoter mutants. Looking at the alkylation pattern in a threedimensional model it appears furthermore that the enzyme recognizes the promoter from just one side of the DNA helix. In a very elegant modification of the methylation protection experiment, Sibenlist succeeded in demonstrating methylation of adenine bases in positions that are accessible to dimethylsulphate only if DNA is melted and in the single-stranded conformation ~7. The region opened upon binding of RNA polymerase, and identified in this way, stretches over about 11 base pairs from the middle of the Pribnow box to just past the RNA start sites ( - 9 to +2, Fig. 2). Which subunits of the enzyme are involved in this process? Photochemical probes linking R N A polymerase subunits to bases in defined positions within the promoter sequence TM revealed that the fl-subunit, which is known to be essential in the catalytic process of RNA synthesis, is cross-linked to the nucleotide in the +3 position of the lac U V 5 promoter, i.e. within the RNA initiation site. The tr-subunit on the other hand was found associated to the - 3 position of the same promoter, i.e. in close proximity to the Pribnow box (Fig. 2). No information was obtained about the positions of any of the

other enzyme subunits and the complete molecular topology of the RNA polymerase bound to promoter awaits further investigation. Kinetics of promoter-polymerase interaction The patterns of RNA synthesis in vitro or in vivo from phage genomes show that different regions are often transcribed with different intensities; the promoters vary in strength. What makes a promoter strong? A priori there are at least three features which may be related to promoter strength: promoter recognition by the enzyme, i.e. the rate of complex formation; the stability of the complex; and the rate of R N A chain initiation. With many promoters the rate of initiation of RNA synthesis is quite rapid compared to complex formation TM. On the other hand complex stability, as well as the rate of complex formation, varies considerably between promoters 5,e.2° and might contribute to the signal strength. When sets of phage promoters differing in their rate of complex formation with R N A polymerase, as well as in the half-lives of the resulting complexes, were analysed with respect to their ability to direct RNA synthesis in vivo and in vitro, a close correlation between the rate of complex formation and the promoter strength was found e. Furthermore the relative rates of complex formation for various E. coli phage and plasmid promoters can differ by factors of 102 to 10 s2°. The promoters that react most rapidly with E. eoli RNA polymerase in vitro are located in the 'early' region of the genome of E. coli phage T5 e. Two of these promoters, which outcompete all others studied so far in polymerase binding and R N A synthesis in vitro, were sequenced (Fig. 3). Both show a prototypic - 1 0 region and contain the highly conserved TTG sequence with the - 3 5 region. In contrast to most other promoter sequences, however, they are extremely A/T-rich (82%) with clusters o f A/T and T/A base pairs, a feature also observed in another promoter which efficiently starts R N A synthesis in vivo~k Whether the high content of AT pairs, and especially the AT block at - 4 3 region, are general indicators of a strong promoter remains to be seen. Since the rate of complex formation with R N A polymerase apparently reflects the signal strength, the absolute values of such rate constants are of interest. In combining some of the published absolute rates with the proper relative rate determinations 2° values are obtained (e.g. for some phage T5 promoters) which suggest that the reaction is faster than a diffusion-controlled

process would allow. Although a direct proof for such fast reaction rates is still pending it is interesting to speculate about mechanisms such as linear diffusion of R N A polymerase along the DNA template, or a direct displacement of the enzyme from unspecific sites.

Concluding remarks Among the various proteins that interact specifically with DNA there are those-that form specific complexes only when certain sequences are provided. The precision involved in the recognition of DNAencoded signals is astonishing. F o r example, the expression of the lac operon is turned off by a repressor protein which is able to recognize its signal - the 'lacoperator', a 27 bp stretch of D N A - among 6 × 108 unspecific sites and most strikingly, 10 repressor molecules per E. coli cell can reduce 1000-fold the expression of that transcriptional unit (for details see Ref. 22). The interaction between bacterial promoters and their respective RNA polymerases is clearly a sequence-specific recognition process: on average R N A polymerase has to identify a promoter among 10 s to 104 unspecific sites. However, unlike the interaction between operator and repressor or between restriction nucleases and their targets, RNA polymerase recognizes a wide range of promoter sequences with differing efficiencies. This in turn modulates the intensity with which various transcriptional units are expressed. It is therefore the individual promoter sequence which defines the signal strength, although we should keep in mind that often the ultimate promoter strength is, in addition, determined by the superimposed action of positive control elements, e.g. the CAP-factor. An 'ideal' or 'generalized' promoter sequence cannot therefore be derived in a straightforward way. One might reduce the problem to 'what does a strong promoter look like?', but even this might turn out to be rather difficult to answer since at least two parameters appear to be involved in the formation of the stable promoterR N A polymerase complex necessary for R N A chain initiation: (a) to be recognized initially by the enzyme a promoter needs a set of precisely positioned bases and there might be several possible arrangements of 'equivalent sets', resulting in sequence variations; (b) during transition from the first contacts to a stable complex at least a part of the promoter undergoes conformational changes. Consequently, base compositions affecting helix stability will also influence

TIBS - October 1980

278 this process. We might therefore envisage promoter families of identical or very similar signal strength but quite different nucleotide sequences. Promoter recognition is certainly a complex biochemical phenomenon and its elucidation still remains a formidable task. As the first step in the controlled flux of genetic information, however, it represents a protein-nucleic acid interaction central to all biological processes and appears, therefore, to be worth a more detailed analysis.

References 1 Burgess, R., Travers, A., Dunn, J. J. and Bautz, E. K. F. (1969)Nature (London) 221, 43-46 2 Zilig, W., Palm, P. and Heil, A. (1976) in RNA Polymerase (Losick, R. and Chamberlin, M., eds), p. 100, Cold Spring Harbor Laboratory 3 Saucier, J.-M. and Wang, J. (1972)Nature (London) New Biol. 239, 167

4 Jones, O. and Berg, P. (1966) J. Mol. Biol. 22, 199 5 Seeburg, P. and Schaller, H. (1975)J. Mol. Biol. 92, 261 6 Gabain, v. A. and Bujard, H. (1977)Mol. Genet. 157,301 7 Schaller, H., Gray, C. and Hermann, R. (1975) Proc. Natl. Acad. Sci. U.S.A. 72,737 8 Schmitz, A. and Galas, D. (1979) Nucleic. Acid Res. 6, 111 9 Pribnow, D. (1975) Proc. Natl. Acad. Sci. U.S.A. 72,784 10 Rosenberg, M. and Court, D. (1979)Annu. Rev. Genet. (in press) 11 Gilbert, W. (1976) in RNA Polymerase (Losick, R. and Chamberlin, M., eds), p. 193, Cold Sping Harbor Laboratory 12 Calos, M. (1978) Nature (London) 274, 762 13 Gilbert, W., Maxam, A. and Mirzabekow, A. (1976) in Control o f Ribosome Synthesis (Kijalgaard, N. and Mal~ae, O., eds), p. 139, Munksgaard, Kopenhagen 14 Sun, L. and Singer (1975) Biochemistry 14, 1795 15 Johnsrud, L. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 5314

16 Siebenlist, U. and Gilbert, W. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 122 17 Siebenlist, U. (1979) Nature (London) 279, 651 18 Simpson, R. B. (1979) Cell 18, 277 19 Chamberlin, M. J. (1976) in RNA Polymerase (Losick, R. and Chamberlin, M., eds), p. 17, Cold Spring Harbor Laboratory 20 Gabain, V. A. and Bujard, H. (1979)Proc. Natl. Acad. Sci. U.S.A. 76, 189 21 Nakamura, Y. and Inoye, R. (1979) Cell 18, 1109 22 Hippel, P. V. (1979) in Biological Regulation and Development (Goldberger, R.F., ed.), p. 279, Plenum Press, New York 23 Smith, B. R. and Schleif, R. (1978)J. Biol. Chem. 253,693 24 Rosenberg, M., Court, D., Shimatake, H., Brady, C. and Wulff, D. L. (1978a) Nature (London) 272,414 25 Pribnow, D. (1975)J. Mol. Biol. 99, 419 26 Post, L., Arfsten, A., Reusser, F. and Nomura, M. (1978) Cell 15,215 27 Dickson, R., Abelson, J., Barnes, W. and Reznikoff, W. S. (1975) Science 182, 27

50 Years Ago E. V. McCollum and oral health Harry G. Day is Professor Emeritus of Chemistry, University of lndiana. He is well known ]'or his work on vitamin A and on trace elements in nutrition, especially fluorine in the prevention of tooth decay. In recent years, he has become a historian of nutrition, especially with respect to the career and influence of E. V. McCollum. T. H. Jukes In October 1930 E. V. McCollum, already widely recognized for his pioneering concepts and basic discoveries in nutrition, was the guest speaker at the New York Academy of Dentistry. He focused on the 'Relationship between diet and dental caries', a subject on which he had been a recognized authority since 1921. McCollum reviewed and evaluated the findings of laboratory and clinical investigators and the observations of other thoughtful persons and directed his attention to knowledge, from whatever source, on the cause of dental decay. In the long discussion that followed, the highly respected Professor L . B . Mendel, who had been McCollum's teacher at Yale 25 years before, aptly stated, 'I have been greatly privileged . . . to have heard presented in a very unusual and stimulating way not only the acquisitions that have come from the recent study of the problems of dental growth and dental disease, b u t . . , some of the problems that need to be considered in the future. '1 In this address and in many other contributions, McCollum advocated searching Elsevier/N0rth-Holland Biomedical Press 1980

inquiry into the cause of dental caries and he marshalled evidence to assail a longprevailing notion that cleaning the teeth is of first importance as a preventive measure to preserve the teeth. As McCollum stated in 1930, 'that idea has been discredited'. From research in his own laboratory beginning in 19212 and extended by others within that decade it was presumed thai the nature of the diet was of great importance in tooth developments and resistance to decay. But the elucidation of the relationships remained elusive. The focus of McCollum in the U.S.A., M. Mellanby in England and others, was on a dietary entity or an imbalance between such entities. As McCollum stated the premise, 'If there is any relation between diet and dental caries, and there is a general belief that there is, it must be explainable, when we have sufficient evidence, on the basis of either a deficiency of one or another of the essential nutrients, or of unfavorable quantitative relations between two or more of them. 'a In 1930the long report by Klein, McCollum, Buckley and Howe a on 'Relation of

diet to the skeletal development of swine, including the development of teeth' provided support for this premise. They showed that the rate of growth is impaired and there are structural defects in the mandibles, incisors and alveolar processes of swine fed on diets unbalanced in calcium and phosphorus. This confirmed and amplified the reports of M. Mellanby in studies on dogs and supported the thesis McCollum first set forth in May 1921 when he spoke to the Iowa State Dental Society~. Based on his general observations and the pioneering studies by Dr C. J. Grieves in McCollum's laboratories on the skulls of rats fed rickets-producing diets, McCollum asserted 'that nutrition investigators, and the dental and medical professions as well, must take a new and broader view of the whole problem of human health, its establishment and its preservation'. It was in 1922, as concluded by G. J. Cox s in 1952, that McCollum, Simmonds, Kinney and Grieves 2 'were the first to record observations of dental caries in the white rat'. The dental decay was produced by restricting the animals to faulty diets. Bunting in 1925 and Marshall in 1927 reported related findings. Also, in 1922, in the second edition of McCollum's The Newer Knowledge of Nutrition, a chapter was included on nutrition and dental health. This was a new departure in books on nutrition. He comprehensively reviewed the theme that: 'the basis of preventive dentistry is satisfactory nutrition during development '6. In each of the three