4
TIBS 14 - August 1989 335 3 Makowski, I., Frolow, F., Saper, M. A., Wittmann, H. G. and Yonath, A. (1987) J. Mol. Biol. 193,819-821 4 Glotz, C., Mfissig, J., Ge~citz, H-S., Makowski, l., Arad, T., Yonath, A. and Winmann, H. G. (1987) Biochem. Int. 15, 953 960 5 Trakhanov, S. D., Yusupov, M M., Agala- rov, S. C., Garber, M. B., Rayazantsev, S. N., Tischenko, S. V. and Shirokov, V. A. (1988) FEBS Lett. 220, 319-322 6 Yonath, A., Glotz, C., Gewitz, H. S., Bartels, K. S., von BOhlen, K., Makowski, I. and Wittmann, H. G. (1988) J. Mol. Biol. 203, 831-834 7 Yonath, A., Frolow, F., Shoham, M., Miissig, J., Makowski, I., Glot;,, C., Jahn, W., Weinstein, S. and Winmann, H. G. (1988) J. Crystal Growth 90, 231 244 8 Yusupov, M. M., Tischenko, S. V., Trakha- nov, S. D., Ryazantsev, S. N., and Garber, M. B. (1988)FEBSLett. 238,113 115 9 Milssig, J., Makowski, 1., von ?36hlen, K., Hansen, H., Bartels, K. S., Wittrnann, H. G. and Yonath A. 11989) J. Mol. Biol. 205, 619~21 10 Klug, A., Holmes, K. C., and Finch. J. T. (1961) J. Mol. Biol. 3, 87-11)1/ 11 Moore, P. B. and Capel M. S. (1988) Annu. Rev. Biochem. Biophys. 17, 34%367 12 Hope, H., Frolow, F., von B6hlen, K., Makowski, I., Kratky, C., Halfon, Y., Danz, H., Webster, P., Barrels, K., Wittmann, H. G. and Yonath, A, (1989) Acta Crystall- ogr. Sect. B 345,190-199 13 Yonath, A., Leonard, K. R. and Wittmann, H. G. (1987) Science236, 813-816 14 Arad, T., Piefke, J., Weinstein, S., Gewitz, H. S., Yonath, A. and Wittmann, H. G. (1987) Biochimie69, 1001 1005 15 Winmann, H.G. (1983) Annu. Rev. Bio- chem. 52, 35~'15 16 Malkin, L. i. and Rich, A. (1967)J. Mol. Biol. 26,329-346 17 Yen, I. J., Macklin, P. S. and Cleavland, D. W. (1988) Nature334, 580-585 18 Bernabeu, C. and Lake, J. A. (1982) Proc. Natl Acad. Sci. USA 79, 3111-3115 19 Milligan, R. A. and Unwin, P. N. T. (1986) Nature 319,693-696 2(1 Wagenknecht, T., Carazo, J. M., Raderma- cher, M. and Frank, J. (1989) Biophys. J. 55, 455-464 21 Gilbert, W. (1963) Cold Spring Harbor Symp. Quant. Biol. 28,287-294 22 Gewitz, H. S., Glotz, C., Piefke, J., Yonath, A. and Wittmann, H. G. (1988) Biochimie 70, 645~548 23 Kurzchalia, S. V., Wiedmann, M., Breter, H., Zimmermann, W., Bauschke, E. and Rapoport, T. A. (1988) Eur. J. Biochem. 172, 663~668 24 Ryabova, L. A., Selivanova, O. M., Bara- nov, V. I., Vasiliev, V. D. and Spirin, A. S. (1988) FEBS Lett. 226,255-260 25 Brimacombe, R., Atmadja, J., Stiege, W. and Schiller, D. (19881 J. Mol. Biol. 199, 115-136 26 Dabbs, E. R. (19861 in: Structure, Function and Genetics of Ribosomes (Hardesty, B. and Kramer, G., eds), pp. 733-748, Springer- Verlag 27 Carazo, J. M., Wagenknecht, T., Raderma- cher, M., Mandiyan, V., Boublik, M. and Frank, J. (1988) J. Mol. Biol. 201,393-404 28 Gewitz, H-S., Glotz, C., Goischke, P., Rom- berg, B., Mfissig, J., Yonath, A. and Win- mann, H. G. (1987) Biochem. Int. 15, 887 895 29 Shevack, A., Gewitz, H-S., Hennemann, B., Yonath, A. and Wittmann, H.G. (19851 FEBS Lett. 184, 68-71 30 Hill, W. E., Trappich, B. E. and Tassanaka- john, B., (19861 in Structure, Function and Genetics of Ribosomes (Hardesty, B. and Kramer, G., eds), pp. 233-252, Springer Verlag How do proteins recognize specific RNA sites? New clues from autogenously regulated ribosomal proteins David E. Draper Some ribosomal proteins which bind specifically to ribosomal RNA also act as translational repressors and recognize their encoding messenger RNAs. The messenger- and ribosomal-RNA binding sites for four of these proteins are now well defined, and striking similarities in primary and secondary structure are apparent in most cases. These 'consensus' structures are useful clues to the features proteins use to recognize specific RNAs. There is a long list of RNA-protein interactions that are known to be important for gene expression and cell function. Many of these interactions appear in the important machinery needed to express proteins (e.g. spliceosomes and ribosomes), while others regulate steps in the pathway from gene to protein. These inter- actions raise the question of how pro- teins recognize specific RNA sites. Are some kinds of RNA structure~ (helices, loops, bulges, mismatches, pseudo- knots) particularly favored as recog- nition features? Do proteins sense specific sequences, particular back- bone conformations, or some combina- D. E. Draper is at the Department o) ° Chemistry, Johns Hopkins Lhm'ersitv, Baltimore. MI) 21218, USA. tion of sequence and structure? To answer these questions the RNA fea- tures recognized by a variety of binding proteins must be compared. However, defining a protein-recognition site in an RNA can be tedious work: the nucleo- tides that form the recognized structure may be dispersed at several locations through a long RNA sequence. A use- ful way to identify such binding sites is to compare two or more RNAs which both bind to the same protein site. Any similarities in sequence or secondary structure then define a 'consensus' site and provide clues to sequences or struc- tures detected by the protein. Finding an RNA consensus site For somc ribosomal proteins consen- sus RNA binding sites can be derived in two different ways. Homologous rRNAs from evolutionarily distant organisms will sometimes bind the same protein; only the conserved fea- tures can be important for recog- nition 1 . Even more informative are the ribosomal proteins which bind both rRNA (in ribosome assembly) and to mRNA (as translational repressors). In these cases two RNA-binding sites with very different functions can be com- pared, and similarities between thcm should be limited to essential features recognized by the protein*. The autoregulation hypothesis The translational repressor activity of some E. coli ribosomal proteins was noticed by several groups about ten years ago 2,3. Most ribosomal protein operons contain one protein which behaves as a repressor: if protein syn- thesis outstrips rRNA synthesis, the accumulating pool of free repressor will bind to the mRNA and repress trans- lation of all the ribosomal proteins in that operon. Translationaily regulated ribosomal protein operons are listed in Table I, along with their identified repressor proteins. In most of these cases the re- pressor protein has been shown specifi- cally to recognize a single target sitc in *The term 'homologous' is restricted to RNAs sharing an evolutionary relationship, while "simi- lar' describes two RNA structures with analogous function but independent origin. (D 198¢,J, Elsevier Science Publishers Lid, (tlK) I)~76 ~(167/g9/$0~.~11

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Page 1: How do proteins recognize specific RNA sites? New clues from autogenously regulated ribosomal proteins

TIBS 14 - August 1989 335

3 Makowski, I., Frolow, F., Saper, M. A., Wittmann, H. G. and Yonath, A. (1987) J. Mol. Biol. 193,819-821

4 Glotz, C., Mfissig, J., Ge~citz, H-S., Makowski, l., Arad, T., Yonath, A. and Winmann, H. G. (1987) Biochem. Int. 15, 953 960

5 Trakhanov, S. D., Yusupov, M M., Agala- rov, S. C., Garber, M. B., Rayazantsev, S. N., Tischenko, S. V. and Shirokov, V. A. (1988) FEBS Lett. 220, 319-322

6 Yonath, A., Glotz, C., Gewitz, H. S., Bartels, K. S., von BOhlen, K., Makowski, I. and Wittmann, H. G. (1988) J. Mol. Biol. 203, 831-834

7 Yonath, A., Frolow, F., Shoham, M., Miissig, J., Makowski, I., Glot;,, C., Jahn, W., Weinstein, S. and Winmann, H. G. (1988) J. Crystal Growth 90, 231 244

8 Yusupov, M. M., Tischenko, S. V., Trakha- nov, S. D., Ryazantsev, S. N., and Garber, M. B. (1988)FEBSLet t . 238,113 115

9 Milssig, J., Makowski, 1., von ?36hlen, K., Hansen, H., Bartels, K. S., Wittrnann, H. G. and Yonath A. 11989) J. Mol. Biol. 205, 619~21

10 Klug, A., Holmes, K. C., and Finch. J. T. (1961) J. Mol. Biol. 3, 87-11)1/

11 Moore, P. B. and Capel M. S. (1988) Annu.

Rev. Biochem. Biophys. 17, 34%367 12 Hope, H., Frolow, F., von B6hlen, K.,

Makowski, I., Kratky, C., Halfon, Y., Danz, H., Webster, P., Barrels, K., Wittmann, H. G. and Yonath, A, (1989) Acta Crystall- ogr. Sect. B 345,190-199

13 Yonath, A., Leonard, K. R. and Wittmann, H. G. (1987) Science236, 813-816

14 Arad, T., Piefke, J., Weinstein, S., Gewitz, H. S., Yonath, A. and Wittmann, H. G. (1987) Biochimie69, 1001 1005

15 Winmann, H.G. (1983) Annu. Rev. Bio- chem. 52, 35~'15

16 Malkin, L. i. and Rich, A. (1967)J. Mol. Biol. 26,329-346

17 Yen, I. J., Macklin, P. S. and Cleavland, D. W. (1988) Nature334, 580-585

18 Bernabeu, C. and Lake, J. A. (1982) Proc. Natl Acad. Sci. USA 79, 3111-3115

19 Milligan, R. A. and Unwin, P. N. T. (1986) Nature 319,693-696

2(1 Wagenknecht, T., Carazo, J. M., Raderma- cher, M. and Frank, J. (1989) Biophys. J. 55, 455-464

21 Gilbert, W. (1963) Cold Spring Harbor Symp. Quant. Biol. 28,287-294

22 Gewitz, H. S., Glotz, C., Piefke, J., Yonath, A. and Wittmann, H. G. (1988) Biochimie 70, 645~548

23 Kurzchalia, S. V., Wiedmann, M., Breter, H., Zimmermann, W., Bauschke, E. and Rapoport, T. A. (1988) Eur. J. Biochem. 172, 663~668

24 Ryabova, L. A., Selivanova, O. M., Bara- nov, V. I., Vasiliev, V. D. and Spirin, A. S. (1988) FEBS Lett. 226,255-260

25 Brimacombe, R., Atmadja, J., Stiege, W. and Schiller, D. (19881 J. Mol. Biol. 199, 115-136

26 Dabbs, E. R. (19861 in: Structure, Function and Genetics o f Ribosomes (Hardesty, B. and Kramer, G., eds), pp. 733-748, Springer- Verlag

27 Carazo, J. M., Wagenknecht, T., Raderma- cher, M., Mandiyan, V., Boublik, M. and Frank, J. (1988) J. Mol. Biol. 201,393-404

28 Gewitz, H-S., Glotz, C., Goischke, P., Rom- berg, B., Mfissig, J., Yonath, A. and Win- mann, H. G. (1987) Biochem. Int. 15, 887 895

29 Shevack, A., Gewitz, H-S., Hennemann, B., Yonath, A. and Wittmann, H.G. (19851 FEBS Lett. 184, 68-71

30 Hill, W. E., Trappich, B. E. and Tassanaka- john, B., (19861 in Structure, Function and Genetics o f Ribosomes (Hardesty, B. and Kramer, G., eds), pp. 233-252, Springer Verlag

How do proteins recognize specific RNA sites? New clues from autogenously regulated ribosomal proteins

David E. Draper

Some ribosomal proteins which bind specifically to ribosomal R N A also act as translational repressors and recognize their encoding messenger RNAs. The messenger- and ribosomal-RNA binding sites for four o f these proteins are now well defined, and striking similarities in primary and secondary structure are apparent in most cases. These 'consensus' structures are useful clues to the features proteins use

to recognize specific RNAs.

There is a long list of RNA-prote in interactions that are known to be important for gene expression and cell function. Many of these interactions appear in the important machinery needed to express proteins (e.g. spliceosomes and ribosomes), while others regulate steps in the pathway from gene to protein. These inter- actions raise the question of how pro- teins recognize specific RNA sites. Are some kinds of RNA structure~ (helices, loops, bulges, mismatches, pseudo- knots) particularly favored as recog- nition features? Do proteins sense specific sequences, particular back- bone conformations, or some combina-

D. E. Draper is at the Department o) ° Chemistry, Johns Hopkins Lhm'ersitv, Baltimore. MI) 21218, USA.

tion of sequence and structure? To answer these questions the R N A fea- tures recognized by a variety of binding proteins must be compared. However , defining a protein-recognition site in an R N A can be tedious work: the nucleo- tides that form the recognized structure may be dispersed at several locations through a long R N A sequence. A use- ful way to identify such binding sites is to compare two or more R N A s which both bind to the same protein site. Any similarities in sequence or secondary structure then define a 'consensus' site and provide clues to sequences or struc- tures detected by the protein.

Finding an RNA consensus site For somc ribosomal proteins consen-

sus RNA binding sites can be derived in

two different ways. H o m o l o g o u s rRNAs from evolutionarily distant organisms will somet imes bind the same protein; only the conserved fea- tures can be important for recog- nition 1 . Even more informative are the ribosomal proteins which bind both r R N A (in ribosome assembly) and to m R N A (as translational repressors). In these cases two RNA-binding sites with very different functions can be com- pared, and similarities between thcm should be limited to essential features recognized by the protein*.

The autoregulation hypothesis The translational repressor activity

of some E. coli ribosomal proteins was noticed by several groups about ten years ago 2,3. Most ribosomal protein operons contain one protein which behaves as a repressor: if protein syn- thesis outstrips rRNA synthesis, the accumulating pool of free repressor will bind to the m R N A and repress trans- lation of all the ribosomal proteins in that operon.

Translationaily regulated ribosomal protein operons are listed in Table I, along with their identified repressor proteins. In most of these cases the re- pressor protein has been shown specifi- cally to recognize a single target sitc in

*The term 'homologous' is restricted to RNAs sharing an evolutionary relationship, while "simi- lar' describes two RNA structures with analogous function but independent origin.

(D 198¢,J, Elsevier Science Publishers Lid, ( t lK) I)~76 ~(167/g9/$0~.~11

Page 2: How do proteins recognize specific RNA sites? New clues from autogenously regulated ribosomal proteins

336 TIBS 14 - August 1989

Tabh, I. Transitionally regulated ribosomal protein operons and their identified repressor proteins"

Number of Operon ribosomal proteins Repressor rRNA sequence name in operon protein binding repressor

u 4 $4 16S 39~400 str 2 $7 16S 926-1391 spc 10 $8 16S 588-651 $20 1 S2(t 16S 39M03 L11 2 L1 23S 2120-2178 S l(t l 1 L4 23S 320-325,615 rif 2 Lll) 23S 1030-1124

"E. coli ribosomal protein operons for which there is both in vivo and in vitro evidence for regulation by a ribosomal p ro te in-mRNA complex are listed. Proteins other than ribosomal proteins are encoded by some of these operons, and there is some evidence for autogenous regulation by proteins S 1, $2 and $63. The approximate rRNA region interacting with each repressor is listed. These have been defined by footprinting and fragment binding experiments, with the exception of L4, for which the only evidence is two prote in-RNA crosslinks.

the mRNA, usually near a ribosome binding site where it presumably inter- feres with ribosome initiation. In multi- gene ribosomal protein operons there is a tendency for a single ribosome to translate all the genes without dissoci- ating from the mRNA. A repressor acting at a single mRNA site may thus repress translation of all the down- stream genes.

The important point for this discus- sion is that each translational repressor recognizcs a specific site in an mRNA. Since all of the proteins identified as repressors also bind directly to the 16S or 23S rRNAs (Table I), it is likely that the mRNAs and rRNAs compete for binding to the same protein recognition site. Any m R N A - r R N A similarities might therefore identify protein-

recognition features. In the last few years the secondary

structures required for translational regulation of a number of the mRNAs listed in Table I have been experimen- tally defined. The secondary structures of rRNA fragments recognizing many of the ribosomal proteins have been characterized for some time 4, and recent work has defined more closely some of these binding sites as well. In this article I compare the mRNA and rRNA binding sites recognized by repressors L1, L10, $8 and $4, which are the best defined systems so far.

Three ribosomal proteins recognize limited RNA structures

For protein LI there is some phylo- genetic information for both the

4o uCAc . 5'UUUUUAUA G Gr~-G~ ~G~C C U C U

I i III I Ill I I I C 60

3' Gu~A UA U- U U A C[CC ~, U~ .~U CLGGAGA

A C UAU A U.80, mRNA L1

63O x

e.a wL 5'A C G A GGIGIAIC UIG C A U G A U/ QUGgA, tt qU

1030 mRNA S8 rRNA

1,~o, ~ , 5'coay c. a q., G,N_Iy '

3'GGCA~uGCGUCCu~AU~C~58O

A U G G G U A

211O-G U ~ 213o A / , ~ / G A~

5'C U U G, U GGUIGG GIGC U U'~ I / / [ I I I l l I I = .....) C CAICC CLCG AG

3'uAAUUu2180 C ~ - - ~ A \ 2 1 6 0

A C rRNA -A c

A U GU

65O I qgA, A, q9 3' U G U U U G 5'

590

1560 1040 ~ 1060

U u u c G c 5 AAGGI~ AGC CA[~ GA , A , . , , . . , , , , . . , , Y G , u Y G , g q q u G

A A A G A A 3, G G C U ~;.~IG(~_ U G G U~L.L'~U A C U A C C G AC GAA - , A [,UI lO8O 111s

mRNA L10 rRNA UGcGAAAG,~

Fig. 1. Comparison o f m R N A (left side) and rRNA (right side) binding sites for ribosomal proteins. Base numberings are from the 5' end o f the E. Coil 16S or 23S rRNA, or the 5' terminus o f the m R N A transcript. 'Tick' marks are located every 10 bases. Boxes outline nucleotides similar in primary and secondary structure between the m R N A and rRNA sites. Underlined bases in the m R N A s indicate the initiator A U G and Shine-Dalgarno sequence. Top, LI binding sites. Bases in boldface are conserved in L I binding sites j?om different bacteria; the sequences shown are J)'om E. coli. Middle, $8 binding sites. Bottom, L8 binding sites.

mRNA and rRNA binding sites. Ll proteins from Serratia rnarcescens and Proteus vulgaris both regulate the E. coli L l l operon 5, and the E. coli LI protein binds an RNA fragment from Dictyostelium discoideurn 26S rRNA which is homologous to the E. coli rRNA binding site~L Homologous nu- cleotides identical in either the three mRNAs or the two rRNAs are shown in bold face in Fig. 1. The conserved bases focus attention on an internal loop in each RNA; the two RNAs have a set of identical bases in the loop and neighboring base pairs (boxed nucleo- tides). In addition, a search for mu- tations which relieve translational regulation in the mRNA turned up point mutations at nearly every nucleo- tide within the box 7, and site-directed mutations within the rRNA box also affect L1 binding ~. In vitro binding experiments with rRNA fragments containing variant sequences or modi- fied bases suggest that the recognition site is confined to the sequences 212(t- 2128 and 2160-2178 (R. A, Zimmer- mann, pers. commun.). All of these results confirm the importance of the boxed nucleotides as L1 recognition features.

An initiating ribosome recognizes the Shine-Dalgarno sequence and initiation codon of the mRNA, and binds to ~30 nucleotides of surround- ing sequence, at about nucleotides 77-107 in the mRNA recognized by El. The boxed L1 recognition site is con- fined to a hairpin just upstream of the ribosome binding site, and it is unlikely that L1 interacts with either the Shine-Dalgarno or initiation codon sequences. The L1- and ribosome- binding sites cannot be completely independent or there would be no translational repression. It is possible that RNA tertiary structure links the two sites together. A hint that this is the case is the observation that a C 4 8 ~ U mutation increases translational ef- ficiency sevenfold 7, even though this nucleotide is outside the expected ribo- some binding site. The effects of L1 binding on the surrounding RNA struc- ture and on ribosome binding arc interesting areas for further work.

The $8 binding site on the 16S rRNA has been very well characterized by chemical protection 9 and genetic m experiments; all indicate the import- ance of an extended hairpin in the rRNA central domain (Fig. 1). This region seems to have an unusual back- bone conformation, as indicated by the weak reactivity of some phosphates

Page 3: How do proteins recognize specific RNA sites? New clues from autogenously regulated ribosomal proteins

T I B S 14 - A u g u s t 1989 337

towards an alkylating reagent 9. On the basis of chemical reactivity exper- iments and model building, a tertiary structure with a U.U base pair and a 'pocket ' of three interacting A residues (shown as bulged bases in Fig. 1) has been proposed; $8 protect',; the same As from reaction with dimethyl- sulfate 9.

Recent site-directed mutagenesis and nuclease protection studies unam- biguously define the mRNA structure shown in Fig. 1 as the target for $8 binding "~'~. There is a striking core of nucleotides identical to the rRNA site, including the three bulged As hypoth- esized to form an unusual tertiary struc- ture. Whether U952 and C955 are single-base bulges in the mRNA is not yet established. They should weaken the helix considerably, and may 'tune' the structure so it is not too stable for ribosomes to disrupt and translate.

The L10 protein associates tightly with a tetramer of another ribosomal protein to form the 'L8' complex, which protects nucleotides 1033-1124 of the 23S rRNA (Fig. 1) from nu- cleases z2. The mRNA translational repression site is unusual in that it is more than 100 bases upstream of the affected ribosome binding site, but its location and secondary structure are supported by L8 nuclease protection studies and site-directed mutagen- esis 13'14. The two RNAs contain two clusters of similar sequences in loops at either end of a six base-pair helix. (Non-Watson-Crick A.C base pairs occur in some tRNA stems, and the one shown at 1050-1109 in the rRNA helix is supported by C---~U changes in some rRNAs.) Helical twist should position the loops on the same side of the helix, where a protein could interact with both.

How L8 binding to the mRNA struc- ture is able to influence translation at the ribosome binding site is an interest- ing question. Mutations near the L8 binding site enhance translation, pre- sumably because they disrupt some long range mRNA structure that affects ribosome initiation L'~. The sec- ondary or tertiary interaction that might link the ribosome and L8 binding sites is unknown.

Common themes in the RNA recognition sites

In all three cases discussed so far the mRNA and rRNA similarities narrow the protein recognition site to a small RNA structure. How precisely do these 'consensus' sites correspond to the

% c u u AqgA,e, " U C I U I U A C - G ® 426 ~ . ~ s ' UGUGCGUUJ~CAU U(~CUGA G-C , , , , , , , , , , , , ,

ACAUGCAAW3GUACGACUUu. . ( 3 . ~ - ~ \ I I I I I U , .~ 'v 3s8 3

u 70 "A U u CC U GA C-G A u 130 -U A C I J - f u

UAAAVAGUAG'GAGUGCAUmRNA 90 AG/ r F I N ~ Fig. 2. Comparison o f the $4 mRNA and rRNA binding sites. The asterisks and underline in the mRNA indicate the Shine- Dalgarno sequence and initiation codons, respectively; 'tick' marks are located every ten nucleotides. Mutations at mRNA base A 106 or compensatory changes in two C. G base pairs weaken $4 binding (circled nucleotides). The phylogenetically conserved secondary structure of the $4 binding region in 16S rRNA is shown; circled regions are similar to mRNA structures. @ is 'footprinted' by $4; changes in the hairpin loop sequence (~) weaken $4 binding; deletion of hairpin (~) does not affect $4 binding.

essential recognition site? A few of the sequence similarities may be coinci- dental: there is about a one in four chance that nucleotides in similar pos- itions in two RNAs will have the same base. Protein interactions may also extend outside the boxed nucleotides to include similar features which do not require identical bases. Either A or G, for instance, can hydrogen bond at the identical N7 position, and proteins can interact with the backbone (there is footprinting evidence for this with $89). Even with these caveats, we expect that the majority of the pro te in-RNA con- tacts take place with the boxed nucleo- tides. The extensive genetic and chemical modification evidence avail- able for L1 and $8 binding sites agrees very well with the 'consensus' sites.

These three protein binding sites are all similar in that they are short helical segments with unusual bulge and loop features; all are likely to have some irregular, unusual tertiary folding. Thus the proteins probably sense the three-dimensional shape of the RNA, as well as the similar sequence features. Helix sequence appears important only at base pairs adjacent to bulges or loops, where the helix is probably dis- torted from its regular conformation. Perhaps base pairs in these positions are more accessible to protein contacts; the A form helix has a deep, narrow major groove which may exclude pro- tein binding.

Other proteins whose RNA binding sites have been defined include the R17 coat protein, which recognizes a 19-base hairpin and is sensitive to sequence changes at only four loop or bulge positions 16, and several

aminoacyl-tRNA synthetases, that recognize two to nine bases distributed throughout the tRNA 17. The consen- sus sites in Fig. 1, which encompass 10-13 bases, suggest that the ribosomal proteins make more sequence-specific contacts than these other proteins. Per- haps ribosomal proteins need an exten- sive set of contacts to hold the RNA in the correct conformation for ribosome assembly or function.

The $4 binding site is a more complex RNA structure

The RNAs recognized by ribosomal protein $4 are much larger than the other three examples. Nuclease protec- tion and footprinting experiments have shown that $4 binds in the 530 base '5'

d o m a i n ' of the 16S rRNA 1s'19, while binding studies with rRNA fragments further limit the recognition site to bases 39-500 (Fig. 2) (J. V. Vartikar and D. E. Draper , submitted). Although footprinting evidence places the $4 binding site near the termini of this large fragment, deletions of hair- pins in the 120-240 region drastically weaken $4 binding (C. K. Tang, J. V. Vartikar and D. E. Draper, unpub- lished). Thus the RNA may have some long range tertiary structure which is required to fold the $4 binding site correctly. The mRNA binding site is limited to a 2130 base fragment and its secondary structure has been proved by site-directed mutagenesis (Fig. 2) 21,22 It is much smaller than the rRNA site, and the RNA is folded in an unusual pseudoknot. (A pseudoknot is an RNA hairpin in which the 3' end extends to base pair with nucleotides in the hairpin loop.)

Page 4: How do proteins recognize specific RNA sites? New clues from autogenously regulated ribosomal proteins

338 T I B S 14 - A u g u s t 1989

The overall secondary structures of the two R N A s are very different. A number of similar sequences in loops and helices can be found, but coinci- dences are possible in R N A s this large. For instance, the most extensive simi- larity, a set of six nearly identical base pairs ( m R N A helix G24--A30/U62- C67 and structure 3 in the r R N A ) , can be altered in ei ther R N A without affec- ting $4 binding 22 (C. K. Tang, J. V. Vart ikar and D. E. Draper , unpub- lished). There are two similarities with experimental support : muta t ions in an A U A loop sequence affect $4 binding in both R N A s (C. K. Tang, J. V. Varti- kar and D. E. Draper , unpublished) and a C G rich helix is footpr in ted by $4 in the r R N A 19 while base-pair changes in a similar m R N A helix also affect $4 affinity 22 (compare structures 1 and 2 in the 16S r R N A and circled bases in the m R N A of Fig. 2). Nei ther of these potential similarities is as impressive as those found in the R N A binding sites of the o ther three proteins discussed.

The weak similarity be tween the R N A s could be explained if $4 uses two different binding sites to bind the two R N A s , but this is not likely. The two R N A s compete for $4 binding, and both the m R N A and the r R N A binding activities show an identical enhance- ment with chloride ion 2°'21. This effect is very unusual, and is evidence that both R N A s are recognized by the same mechanism. The weak similarity is more likely a result of $4 recognizing some tertiary structure which is com-

mon to the R N A s but not evident at the level of secondary structure. The r R N A , with its junctions of helices and potential for extensive tertiary inter- actions, could create a binding site composed of several different helix and loop segments brought together in a particular three-dimensional configur- ation, The unusual m R N A pseudokno t folding, which brings several helical segments close together , may be a way of creating the same essential three- dimensional a r rangement of R N A strands in a smaller structure. Thus the $4 recognit ion features could be widely separated in a two dimensional drawing of the secondary structure and difficult to discern as similarities. As the $4 recognit ion features are mapped in the two R N A s , clues to the three dimen- sional organizat ions of complex R N A structures may be obtained.

Acknowledgements W o r k f rom the author ' s lab has been

suppor ted by N I H grant GM29048 and a Research Career Deve lopmen t Award (CA01081).

References 1 EI-Baradi, T. T. A. L., de Regt, V. C. H. F.,

Einerhand, S. W. C., Teixido, J., Planta, R. J., Ballesta, J. P. G. and Rau6, H. A. (1987) J. Mol. Biol. 195,909-917

2 Nomura, M., Gourse, R. and Baughman, G. (19841 Annu. Rev. Biochem. 53, 75-118

3 Lindahl, L. and Zengel, J. M. (1986) Annu. Rev. Genet. 2(1,297-326

4 Noller, H. F. (1984)Annu. Rev. Biochem. 53, 118-162

5 Sot, F. and Nomura, M. (1987) Mol. Gen. Genet. 21(I, 52-59

6 Gourse, R. L., Thurlow, D. L., Gerbi, S. A. and Zimmermann, R. A. (1981) Proc. Natl Acad. Sci. USA 78, 2722-2726

7 Thomas, M. S. and Nomura, M. (1987) Nu- cleic Acids Res. 15,3085-3096

8 Said, B., Coles, J. R. and Nomura, M. (1988) Nucleic Acids Res. 16, 10529-10545

9 Mougel, M., Eyermann, F., Westhof, E., Romby P., Expert-Bezanqon, A., Ebel, J-P., Ehresmann, B. and Ehresmann, C. (1987) J. Mol. Biol. 198,91-107

10 Gregory, R. J., Cahill, P. B. F., Thurlow, D.L. and Zimmermann, R. A. (1988) J. Mol. Biol. 204,295-307

11 Ceretti, D. P., Mattheakis, L. C., Kearney, K. R., Vu, L. and Nomura, M. (1988) J. Mol. Biol. 204,309-329

12 Beauclerk, A. A. D., Cundliffe, E. and Dijk, J. (1984)J. Biol,. Chem. 259, 6559-6563

13 Johnsen, M., Christensen, T., Dennis, P.P. and Fill, N. P. (1982) EMBOJ. 1,999-1004-

14 Climie, S. and Friesen, J. D. (1987) J. Mol. Bio1197,371-381

15 Fill, N. P., Friesen, J. D., Downing, W. L. and Dennis, P. P. (1980) Cell 19,837-844

16 Romaniuk, P. J., Lowary, P., Wu, H-N., Stormo, G. and Uhlenbeck, O. C (19871 Bio- chemistry 26,156,3-1568

17 Schulman, L. H. and Abelson, J. (1988) Science 240, 1591-1592

18 Ehresmann, C., Stiegler, P., Carbon, P., Ungewickell, E. and Garret, R. A. (19801 Eur. J. Biochem, 103,439-446

19 Stern, S., Wilson, R. C. and Noller, H. F. (1986)J. Mol. Biol. 192,101-110

20 Vartikar, J. V. and Draper, D. E. J. Mol. Biol. (in press)

21 Deckman, I. C., Draper, D. E. and Thomas, M. S. (1987) J. Mol. Biol. 196,313-322

22 Tang, C. K. and Draper, D. E. (1989) Cell57, 531-536

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