THE JOURNAL OF BIOLOGICAL

Minireview Vol. 264?No.4, Issueof.Feb

CHEMISTRY
1903-1906,1989
8 1989 by The American Society of B i o c h e n u n t r y ~ ~ o ~ &Biol
3.2
Printsd

The Helix-Turn-HelixDNA
Binding Motif
Richard G . Brennan and Brian W.Matthews
From the Institute of Molecular BWloBy and Department of
Physics, University of Oregon, Eugene, Oregon97403

One of the most striking findings to emerge from the determina-

tions of the structures of Cro, CAP, and X-repressor (1-3) was that
these three sequence-specificDNA-binding proteins had in common
a substructure now named the helix-turn-helix motif. The apparent
importance of this motif was suggested not only by its striking
similarity in therespective structures (4,5) but also from amino acid
sequence comparisons (6-11) which indicated that a similar motif
might occur in other DNA-biding proteins.
It was proposed (1,3, 12-14) that thehelix-turn-helix motif inter-
acts with operator DNA in a manner simiiar to that shown in Fig. 1
for Cro. Recognition of a specific sequence on DNA was thought to
be achieved bya network of hydrogen bonds and other contacts FIG. 1. Model for the presumed complex of a dimer of Cro
between the side chains of the protein and the parts of the base pairs repressor with DNA (1, 12). For clarity the side chains of the
exposed within the grooves of the DNA. The second helix of the residues within the helix-turn-helix units have been removed, and
helix-turn-helix motif, which was thought to bind withii the major only the a-carbon atomsare shown as small white spheres. The helix-
groove of the DNA,was presumed to be especially important in turn-helix on the right of the figure is most obvious. The first helix
recognition. extends from right to left; the second helix (the “recognition helix”)
The importance of the helix-turn-helix motif in DNA-protein is seen end-on within the major groove of the DNA. For the remainder
of the protein acidic residues are drawn red, basic residues blue,
interaction has been supported by awealth of biochemical and genetic hydrophilic noncharged residues are green, and hydrophobic residues
evidence (e.g. see Refs. 15-18) and has now been shown directly by are shown gray (original figure prepared by Dr. D. H. Ohlendorf,
recent structure determinations of a number of complexesof repressor University of Oregon and Du Pont).
proteins with synthetic operators. These include the DNA-binding
domains of the repressors of phage 434 (19,20) and phage X (21), Cro “master set” consists of 10 proteins (Fig. 2). All but three of these.
protein from phage 434 (22), and trp repressor from Escherichia coli (P22 Cro, P22 repressor (cII), and X cII protein) are now confirmed
(23). In each case the helix-turn-helix motif is an integral part of theas containing a helix-turn-helix motif (see above). An amino acid
protein-DNA interface with the second a-helix (the “recognition sequence of interest is compared with the master set by evaluating,
helix”) located within the major groove of the DNA (cf. Fig. 1). in turn, the correspondence between every possible 22-amino acid
The purpose of this report is notto review, in detail, the structuressegment and the master set of sequences. For largely historical
of these repressor-operator complexes. Rather, we will first discuss reasons it is convenient to use a system of scoring in which sequences
the use of amino acid sequence comparisons to locate putative helix- with the best correspondence with the master set have the lowest
turn-helix motifs. Then we will review the structural correspondence scores. The score for each 22-amino acid segment is determined by
between known helii-turn-helix units. Finally, the role of the helii- summing the total number of amino acids in the master set thatdiffer
turn-helix in recognition of sequence-specific binding sites on DNA with the segment being tested. The score so obtained is normalized
will be evaluated in light of the recently defined repressor-operator by dividing by 220 (Le. 22 amino acids X 10 sequences in themaster
complexes. set) to give the average amino acid change per codon (AAC). Scores
obtained from comparing many 22-amino acid segments with the
Sequence Comparisons master set are pooled to provide statistical estimates of the signifi-
There has been considerable interest in the use of amino acid cance of an individual AAC score (9,31).
sequences to identify putative helix-turn-helix motifs in proteins of Segments with the lowest scores (AAC values) are themost likely
unknown structure. Predictions that have been confirmed by subse- candidates as helix-turn-helix motifs. It is not possible to draw a
quent structure determination include the helix-turn-helix units of sharp dividing line between those proteinsthat do have a helix-turn-
X-repressor (3, 6), trp repressor (9, 23, 24), 434 Cro protein, and 434 helix and those that probably do not. If, however, a 22-amino acid
repressor (6, 7, 19,22, 25, 26). Prediction of a helix-turn-helix in loc segment is found with an AAC score of 0.80 or less, it can be regarded
repressor (7,8)has been verified by two-dimensional NMR spectros- as a strong candidate for a helix-turn-helix motif. Such sequences are
copy (27), and predictions for the biotin repressor of E. coli (28) are shown in Fig. 2.’
strongly supported by genetic analysis (29). This procedure successfully predicts all known examples of helii-
These successful predictions give confidence in the methods on turn-helix motifs in sequence-specific DNA-binding proteins. In ad-
which they were based. They also provide a representative set of dition it does not misidentify any 22-amino acid segments that are
examples against which new predictions can be assessed. known not to be helix-turn-helix units.
In comparing amino acid sequences, especially those that have An amino acid sequence that corresponds to a putative helix-turn-
marginal correspondence, it is essential that defined statistical pro- helix motif can be given greater credence if it is compatible with
cedures be used. Also it is very desirable that the significance of a known stereochemistry (9). Inspection of the structure of Cro sug-
proposed matching of sequences be assessed by controls carried out gested that a helii-turn-helix unit would probably have the following
with unrelated or jumbled sequences (e.g. see Refs.8,30, and31). We characteristics, with residue numbering as in Fig. 2 (9). (i) Residue 9
will focus our discussion on the method employed in our laboratory should be a glycine(having a conformation rare for nonglycines). (ii)
to search for putative helix-turn-helix units and, as listed above, Residues 4 and 15 should not be charged (these residues being buried
found to be effective in a number of cases.
A new amino acid sequence is compared with a “master set” of Tables giving the AAC scores for these and other DNA-binding
prealigned sequences, 22 amino acids in length, taken from proteins proteins, as well as statistical estimates of significance and a full set
known or assumed to contain helix-turn-helix units (9, 31). Our of references, are available on request.

1903
1904 Minireview: Helix-Turn-Helix DNA Binding Motif
k- H e l i x --+Turn +- H e l i=x= - . Score

- 2 -1 1 2 3 4 5 6 7 8 9 10 11 1 1
23 14 11
5167 18 1 9 20

M a s t e rs e t of s e q u e n c e s

X Cro 1 4 - F G Q T K T 4 K D L C V Y Q S A J N K A I H 0.70

434 C r o 1 6 - M T Q T E L A T K A G V K Q Q S l Q L I E A 0.66

P22 Cro 1 1 - G T Q R A V A K A L G I S D A A q S Q n K E 0.67

X Repressor 31-L S Q E S V _A D K M _C M G Q S G 1 G A L F N 0.70

434 Rep. 1 6 - L N Q A E L A Q K V G T T Q Q S L E Q L E N 0.65

P 2 2 R e p . ( C129)- 1 R Q A A L G K M V 6 V S N V A I S Q W E R 0.67

X C I I 2 4 - L G T E K T _ A E A V _ G V D K S Q J S R W K R 0.75

lacR 4 - V T L Y D V A E Y A G V S Y Q T P S R V V N 0.69

CAP 1 6 7 - 1 T R Q E I G Q I V G C S R E T ~ G R I L K 0.70

trp R 6 6 - M S Q R E L K N E L G A G I A T L T R G S N 0.71

Other DNA-bindinn proteins

C l P22 23-R C Q R K V A D A L C I N E S Q I S R W K G 0.71

GalR 2 -A T I K D V A R L A G V S V A T P S R V I N 0.73

FIG. 2. Amino acid segments that T7 gene 7 . 1 0 2 - L S H R S L G E L Y G V S Q S T L T R I L Q 0.73

agree best with a master set of 10
helix-turn-helix motifs. The score TetR 25-L T T R K L A Q K L G V E Q P T L Y W H V K 0.75
(AAC value) gives the correspondence
T7 g e n e 1 4 . 0 119-D Y Q A I F A Q Q L G G T Q S A A S Q I D E 0.76
between each segment and the master
set (lower values indicate better agree- DeoR 22-L H L K D A A A L L G V S E M T I R R D L N 0.76
ment). The number to the left of each
segment is the amino acid sequence <J4 330-R T L E E V C K V F C V T R E R J R Q I E A 0.76
number of the first residue in the seg-
1’7 g e n 2e . 1
80 9 - E S N V S L & R T Y G V S Q Q T L C D T R K 0.76
ment. The numbering at the top of the
figure follows the convention of Pabo I m m Rep (1
4 )7 - 5 T L E A V q G A L G I Q V S A I V G E E T 0.77
and Sauer (17). Amino acids that are
most strongly conserved (Ala or Gly in Frlr 195“ T R C D I C N Y L G L T V E T l S R L L G 0.78
position 5, Gly in position 9, and Ile or
MerR ( 1 ) 8-L T I G V F A K A h G V N V E T L R F Y N R 0.78
Val in position 15) are underlined.
I m m Rep ( 2 ) 18-L T Q V Q L A E K A N L S R S Y L A D I E R 0.79

0 32 2 5 1 - S T L Q L E _ A D R Y _ G V S A E R Y R Q L E K 0.79

Leu0 1 8 - Q N I T R A A H V L G M S Q P A Y S N A V A 0.79

LysR 1 9 - G S L T E A A H L L H T S Q P T Y S R E L A 0.79

AmpR 2 1 - L S F T H A A I E L N V T H S A I S Q H V K 0.80

AntP 9 1 4 3 P Q A Q T N G Q L G V P Q Q Q Q Q Q Q Q Q 0.80

Nul X 3 ” J N K K Q L _ A D I F C A S I R T J Q N w Q E 0.80

MuB 1 9 - T T F K Q 1 4 L E S p L S T G T L S S F I N 0.80

d n a B ( E . c o l i ) 366-R S L K A L A K E L N V P V V A L S Q L N K 0.80

BirA 2 0 - H S G E Q L G E T L G M S R A A L N K H I Q 0.80

T7 g e 1n 8e . 5 61-K Y Q E D L A A L E G T S D R I 1 S D I. R S 0.80

DBPIl (Rm) 5-E L V A A V A D K A G L S K A D A S S A V D 0.80

CysB 1 7 - L N V S S T & E G L Y T S Q P G L S K Q V R 0.80

CytR l o - A M I K D V A L K A K V S T A T E S R A L M 0.80

MaLal 1 1 4 - K E K E E V A K K C G I T P L Q 1 R V W V C 0.80

P2R 1 8 - L S R Q Q L A D L T G V P Y G T L S Y Y E S 0.80

in Cro). (iii) Residues 3-8 and 15-20 should not be proline (being
within a-helices). (iv) Residue 5 should not be a @-branchedresidue
(being wedged between the two helices).
These characteristics are not absolute, and itis quitepossible that
they could be violated by a bona fide helix-turn-helix motif (e.g. a
functional mutant of A-repressor lacks the “invariant” glycine at
position 9 (34)). However, it is worth noting that none of the most
strongly predicted sequences in Fig. 2 (AAC < 0.79) violates these
expected characteristics.
There are anumber of proteins that have been proposed to include
potential helix-turn-helix motifs but agree poorly with the master set
of reference sequences used here. Examples include homeo boxes,
histones, some transcriptional activator proteins, resolvases and in-
version proteins, and the AIDS protein “tat.”’ The poor scores asso-
 Minireview: Helix-Turn-Helix DNA Binding Motif
ciated with these proteins do not, of course, prove that they do not
have helix-turn-helix motifs; it only suggests that the likelihood is
not high.
Dodd and Egan (32) have suggested a method of predicting putative
helix-turn-helix motifs that is similar in principle to theone described
here. It uses a masterset of 37 sequences and a somewhat complicated
method of scoring agreements. On the whole it gives results similar
to those shown in Fig. 2, although it does not successfully predict the
known helix-turn-helix motif in trp repressor. Another method of
predicting helix-turn-helix motifs has been proposed by White (33).
This method also does not predict the known helix-turn-helix in trp
repressor and, inaddition, incorrectly suggests helix-turn-helix motifs
in myoglobin and superoxide dismutase.
Structural Comparisons
The initial recognition of the significance of the helix-turn-helix
motif derived as much from structural comparisons as from consid-
erations of amino acid sequences. In particular, CAP and Cro were
seen to contain a similar helix-turn-helix substructure in their pre-
sumed DNA binding regions even though the rest of these protein
structures had little in common (4). 24 a-carbon atoms (residues 13-
36) including the helix-turn-helix of Cro could be superimposed with
A
a root mean square discrepancy of 1.1 on the corresponding 24 a-
carbons (166-189) of CAP (4). The significance of this result was
enhanced by the finding that the structure of X-repressor also con-
tained a helix-turn-helix substructure (5). In this case 23 a-carbons
(residues 31-53) could be superimposed on residues 14-36 of Cro with
a root mean square discrepancy of only 0.7 A.
A systematic search
through all protein structures in the Brookhaven Data Bank failed
to find any 22 a-carbon segment that corresponded to thehelix-turn-
helix as seen in Cro, X-repressor, and CAP (4,35) (Fig. 3).
Now that the helix-turn-helix has been observed in a number of
other DNA-binding proteins, it is possible to extend the initial
structural comparisons. The correspondence between the helix-turn-
helix unit of Cro (1) with those seen in CAP (2), X-repressor (3), trp
repressor (24), phage 434 Cro (25), and phage 434 headpiece (26) is
given in Table I. In making these comparisons Cro was chosen as the
“reference” structure, in part because it was used for the initial
comparisons, but also because it agrees better with most of the other
helix-turn-helix motifs than they do among themselves. The back-
bone segment that is most precisely conserved in all six of the above
proteins consistsof 21 a-carbon atoms (Table I). Outside this region,
the individual structures may differ substantially.
There are three other proteins that have been reported to contain
I
15
R.rn.s. dlfferencs (dl
FIG. 3. Histogram showing the result of a search through
the Brookhaven Data Bank for helix-turn-helix motifs. The
inset shows the superposition of the helix-turn-helix motifs as initially
seen in Cro and CAP (4). The horizontal axis is the root-mean-square
discrepancy between the 24 a-carbon atoms in the helix-turn-helix
of Cro and all possible 24 a-carbon segments for the known protein
structures. Also shown are the root mean square discrepancies be-
tween the helix-turn-helix of Cro and the eight other proteins listed
in Table I. Note that thehistogram is taken from the original search
(4,34) and was carried out using the 24 a-carbon atoms 13-36 of Cro
repressor. In contrast, the individual values 1-8 shown in the figure
were calculated for the shorter21 a-carbon segment 16-36. As is clear
from the figure and from Table I, this 21 a-carbon segment is well
conserved in all known repressor and activator crystal structures.
Beyond the 21 a-carbons, however, the protein backbones may differ
substantially in the different structures.
1905
TABLE I
Structural correspondence between helix-turn-helix motifs
The table gives the root mean square discrepancy between 21 a-
carbons in the helix-turn-helix of the subject protein and thatof Cro
repressor. The rank corresponds to the numbering used in Fig. 3.
Protein Residues Discrepancy Rank
A
X Cro 16-36
Phage 434 repressor 17-37
1 0.43
Phage 434 Cro 19-39 0.56 2
X repressor 33-53
3 0.68
Trp repressor 64-84 0.89 4
CAP 169-189 0.90 5
Cytochrome c peroxidase 132-152 1.39 6
DNA binding protein I1 7-27 1.43 7
L7/L12 ribosomal protein 16-36 1.61 8
substructures similar to thehelix-turn-helix motif. The first of these
is "DNA-binding protein 11,” a nonspecific DNA-bindingprotein from
Bacillus stearothermophilus (36), the second is cytochrome c peroxi-
dase, and the third is the ribosomal protein L7/L12 (Table I). The
latter two helix-turn-helix structures were found in a recent search
through the Brookhaven Data Bank by Richardson and Richardson
(37). Although the helix-turn-helix units inthesethreeproteins
correspond reasonably well with that of Cro, none agrees as well as
the helix-turn-helix units in the proteins that are known to bind
DNA sequence specifically (Fig. 3). Nevertheless, it is intriguing that
two of the three examples are proteins that interact with nucleic
acids. The amino acid sequences within these three helix-turn-helix
units do not obviously correspond with the master set of 10 sequences.
However, the amino acid sequences of several homologs of the non-
specific DNA-binding protein have been determined and one of these,
from Rhizobium melitoti (DBPZZ ( R m ) )yields a score of 0.80(Fig. 2).
Furthermore, this score is for the set of 22 amino acids that corre-
spond to the observed helix-turn-helix in the B. stearothermophilus
enzyme. The significance, if any, of this correspondence is a matter
for debate.
Role of the Helix-Turn-Helix in Recognition
For Cro protein the “recognition helix” (i.e.the second helix of the
helix-turn-helix motif) protrudes from the surface of the protein and
matches in shapethe major groove of the DNA (Fig. 1). This comple-
mentarityinshapecan facilitate binding of the protein to both
sequence-specific and nonspecific sites on the DNA.As shown in
Table I, the backbone conformations of the helix-turn-helix motifs
in other sequence-specific DNA-binding proteins are very similar to
that seen in Cro. Notwithstanding this overall similarity, the helix-
turn-helix motifs do not interact with the DNA in exactly the same
way in each repressor-operator complex (19-23). This had been
anticipated from early structural comparisons of Cro and X-repressor
(5, 13), although “helix-swap” experiments (38,39) suggested similar
rather than dissimilar geometries of binding. It can nowbe seen
directly in the different repressor-operator complexes that the re-
spective helix-turn-helix motifs adopt approximately similar but dis-
tinctly different binding geometries with respect to theDNA operator
(19-23).
The helix-turn-helix does not function as a fixed “reading head”
that is always aligned in the same way relative to the DNA. Rather,
recognition is achieved by a combination of factors. Specific hydrogen
bonds and van der Waals contacts between the side chains of the
protein and the partsof the base pairs exposed within the grooves of
the DNA clearly play a major role. Appropriately placed water mole-
cules that bridge between the protein and the DNA may also partic-
ipate in the recognition of a specific sequence. The DNA may also
adapt to enhance contacts within the grooves as well as interactions
with the phosphate backbone.
The role of the helix-turn-helix appears to be to provide a rigid
underlying framework that supports the recognition surface of the
protein but not to define how that recognition surface is aligned with
the DNA.
The complexes involving X-repressor headpiece, 434 Cro, and 434
repressor headpiece all support the expectation that these proteins
recognize their specific binding sites on the DNA by direct contacts
with the edges of the base pairs exposed within the grooves of the
DNA (19-22). In the complex of trp repressor, however, there are no
1906 Minireview: Helix-Turn-Helix DNA Binding Motif
direct contacts between the protein and the base pairs that have been
shown in uiuo to be important in recognition. Sigler and colleagues
(23) argue that trp repressor provides an example of indirect readout.
In other words, they suggest that trp operator is recognized indirectly
through sequence-specific changes induced in the geometry of the
phosphate backbone that, in turn, permit the formation of a tight
complex with the protein. This rationalization for the specific binding
of trp repressor poses several questions. In particular, the observed
distortions in the structure of the trp operator are notlarge, and it is
not obvious why different DNA sequences could not assume confor-
mations similar to that adopted by the trp operator. The crystals of
the trp repressor-operator complex were grown from 50 mM NaC1, 11
mM CaCL, 10 mM cadodylate and 35% methylpentanediol(23). Such
low salt, high alcohol conditions would be expected to increase the
affinity of nonspecific DNA binding. Indeed, von Hippel and co-
workers (40) have shown that in low salt the affinity of lac repressor
for nonspecific sites on DNA can approach, if not exceed, its affinity
for the lac operator. Furthermore, they have also shown that the
nonspecific binding of lac repressor is enhanced by the presence of
glycerol (41). If trp repressor behaves similarly to lac repressor, it
raises the possibility, first suggested to us by von Hippel,' that the
conditions necessary to crystallize the trp repressor-operator complex
may have resulted in a sequence nonspecific complex. This question
will need to be addressed in future studies.
The reason for the precise conservation of the backbone configu-
ration of the helix-turn-helix motif in the different repressors is not
obvious. If different helix-turn-helix motifs were required to align on
the DNA in exactly the same manner, then it would be easy to argue
that this required strict conservation of the shape of the helix-turn-
helix motif. As was noted, this is not the case. Different helix-turn-
helix motifs are seen to bind to DNA with different alignments.
Recent evidence (42-44) suggests that thehelix-turn-helix unit of lac
repressor could be aligned on the DNA in adirection opposite to that
of X-repressor, cro 434, and trp repressor.
There is also no obvious structural reason for the conservation of
the helix-turn-helix motif. Because structures corresponding to the
helix-turn-helix motif are not found in protein structures in general
(Fig. 3), itseems unlikely that thehelix-turn-helix unit has a confor-
mation that is energetically favored during the folding process. In-
spection of observed helix-turn-helix motifs shows that there can be
direct van der Waals contactsbetween residues in the "elbow" region,
but the area in contact isrelatively small. Pabo and Sauer (17) have
noted that the contact residues include the highly conserved alanine
at position 5 and the conserved isoleucine or valine at position 15
(Fig. 2) and have suggested that this"invariant" contact helps main-
tain the angle between the two a-helices. On the other hand, the
observations that CAP has a glycine and trp repressor has a lysine in
place of the critical alanine suggest that the interhelix angle is not
determined by these contacts alone.
Perhaps the most reasonable explanation is that the conservation
of the backbone of the helix-turn-helix motif is anatural consequence
of evolution. Presumably all helix-turn-helix motifs that have amino
acid sequence correspondence evolved from a common precursor. It
is well known that the amino acid sequence of a protein changes
much more rapidly during evolution than does its three-dimensional
structure. For this reason, the backbone of the helix-turn-helix motif
would be expected to be conserved during evolution. If, in addition,
the helix-turn-helix were to be intimately associated with the DNA
in the DNA-protein complex, then it would be heavily constrained
not to change its shape. In this scenario, the evolution of DNA-
binding proteins specific for different operators would be achieved by
a sequential process in which changes of individual amino acids within
the helix-turn-helix motif, as well as larger changes elsewhere in the
DNA-binding protein, could result in new sequence preferences, but
the backbone of the helix-turn-helix motif would remain essentially
invariant.
' P. von Hippel, personal communication.
Acknowledgments-We thank Dr. Peter von Hippel for helpful
discussions on sequence-specificand sequence-nonspecificDNA-pro-
teininteraction. We are also grateful to Drs. C. 0. Pabo, S. C.
Harrison, P. B. Sigler, and T. A. Steitz for providing coordinates, and
Drs. J. S. Richardson and D.C. Richardson for a copy of their
manuscript in advance of publication.
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