The EMBO Journal vol.3 no. I pp.
121 - 126, 1984
The binding of 6-demethylchlortetracycline to 70S, 50S and 30S
ribosomal particles: a quantitative study by fluorescence anisotropy
Bernd Epel* and Paul Woolley
Max-Planck-Institut fur Molekulare Genetik (Abt. Wittmann), Ihnestrasse
63, D-1000 Berlin (West) 33
'Present address: Institut fur Toxikologie, Versbacherstrasse 9, D-87
Wurzburg, FRG
*To whom reprint requests should be sent
Communicated by R. Garrett
The binding of demeclocycline (6-demethylchlortetracycline)
to ribosomes and ribosomal subunits from Escherichia coli
was investigated by using the fluorescence anisotropy of the
antibiotic to determine the extent of binding. Binding data
obtained from 70S and 30S particles differed fundamentally
from those obtained from 50S subunits: the first two showed
a strong, specific interaction while the third did not. In addi-
tion, all three particles possessed weak, unspecific binding
sites. Computer-aided least-squares analysis of the data
yielded the following numbers of sites and equilibrium
constants: for 30S, n, = 1, K1 = 2.2 x 106 M-1, n2 K2 =
0.029 x 106 M-1; for 50S, n, = 0, n2K2 = 0.035 x 106 M1;
for 70S, n1 = 1, K1 = 3.2 x106M-1, n2K2 = 0.082 x106
M- 1. These data resolve current disagreement in the
literature and are a prerequisite for quantitative studies of the
mechanism of inhibition by tetracycline of protein bio-
synthesis.
Key words: ribosome/tetracycline/fluorescence/anisotropy/
antibiotic
Introduction
After certain penicillins, the tetracyclines are the most
frequently-used broad-spectrum antibiotics. It is generally ac-
cepted that the main target of their action is the ribosome,
where they interfere with the binding of aminoacyl-tRNA in-
to the ribosomal A (acceptor) site, thus inhibiting protein syn-
thesis (Suarez and Nathans, 1965; Sarkar and Thach, 1968).
In addition to their application in therapy, the tetracyclines
have frequently been used to block the ribosomal A site in
functional tests. In spite of the clinical and biochemical im-
portance of tetracyclines, little is known about the mode of
their interaction with the ribosome. Their biological activity
has been ascribed variously to multiple, weak interactions
(Fey et al., 1973) and to single, strong interactions (Tritton,
1977; Streltsov et al., 1974, 1975). Several efforts have been
made to determine, by various methods, at least the number
and the strength of the ribosomal binding sites for tetra-
cycline, but the results reported appear to differ widely (Table
I).
Although the general trend of evidence seems to support
the assertion that the activity of tetracyclines in inhibiting
protein synthesis is associated with a single, specific binding
site (reviewed by Gale et al., 1981) on the ribosome, this site
has been claimed variously to be found on 70S and on 30S
particles (Connamacher and Mandel, 1965, 1968), on both
30S and 50S particles (Day, 1966a, 1966b) and on 70S par-
Oc IRL Press Limited, Oxford, England.
ticles only (Werner et al., 1975). The wide-ranging disagree-
ment may be due to experimental problems such as the
limited stability of many tetracyclines, which makes long-time
methods such as equilibrium dialysis unreliable, the presence
of weak binding sites, which demands exact and numerous
data-points, and the use of techniques which involve physical
separation (e.g., nitrocellulose filtration) and thus risk
displacing the binding equilibrium.
We describe here the use of fluorescence anisotropy to
determine the degree of binding of demethylchlortetracycline
(demeclocycline INN) to ribosomes and ribosomal subunits.
The tetracycline derivative chosen combines high biological
activity (150% compared with unmodified tetracycline;
Brown and Ireland, 1978) with high stability in solution and it
is strongly fluorescent. Anisotropy measurement is a techni-
que particularly suitable for monitoring the extent of tetra-
cycline-ribosome binding, since the mobility of the molecule
is reflected in its anisotropy, which rises when the small
fluorophore becomes bound to the much larger ribosomal
particle. The method is rapid, so that enough data-points can
be collected in a reasonable time, thus reducing uncertainty
due to the deterioration of the biological material; it is direct,
so that physical separation is obviated; it is exact, since it is an
intensive quantity and is not influenced by small variations in
the sample concentration.
This work establishes the binding pattern of one strong and
many weak sites, with the strong binding site located (in
agreement with the results of photo-affinity labelling ex-
periments - see Discussion) on the 30S subunit. These data
are a pre-requisite for the investigation of the mechanism of
tetracycline action at the thermodynamic level, to be describ-
ed in a later publication.
Results
Fluorescence characteristics of demeclocycline
Figure 1 shows the absorption, excitation and emission spec-
tra of free demeclocycline dissolved in buffer at 5 mM
magnesium at 25°C. The binding constant for magnesium
and demeclocycline was estimated by fluorescence titration
under these conditions to be 4900 i 250 M- 1, so the spectra
represent the complexed form (the uncomplexed demeclo-
cycline ligand does not contribute appreciably to the fluores-
cence). The quantum yield was determined as 2.2% and the
anisotropy as 0.121 4 0.005 at 25°C. Since demeclocycline
has a mol. wt. of 501, the fluorescence lifetime is therefore
-
0.3 ns or less; this relatively short lifetime is consistent with
the low quantum yield.
Binding of demeclocycline to ribosomes shifts the emission
maximum only slightly ( <4 nm) to longer wavelength. After
an equilibration time of 15 min the anisotropy values of free
and bound demeclocycline are constant at 250C for several
hours. A very small decrease in intensity during irradiation
was observed (<0.20/o during the acquisition of a spectrum).
The anisotropy of bound antibiotic was calculated in-
121
B. Epe and P. Woolley
Table I. Equilibrium constants reported for tetracycline-70S interactions at 10 mM magnesium
Method First (strong) binding
K1 (M- 1)
Equilibrium dialysis 1.29 x 106
Filter assay; concentration deter- 4-6 x 10
mined by fluorescence intensity
Two-phase partitioning 0.5-2.5 x 108
Equilibrium microdialysis 8.5 x 106
aStreltsov et al., 1974, 1975.
bTritton, 1977.
cLe Goffic et al., 1980.
dKozak and Zelinka, 1980.
300 400 500 600
Wavelength (nm)
Fig. 1. Absorption ( ) and fluorescence (--- -) spectra of
demeclocycline in the presence of magnesium (5 mM). The absorption
spectrum is shown in units of (mM)- 1 cm- 1 (Xu = 378 mm,
E378 = 19 200 M- 1 cm 1) and the fluorescence spectra (left, quantum-
corrected excitation; right, uncorrected emission) in arbitrary units. The
difference between the wavelengths of the absorption and excitation max-
ima is attributed to experimental error.
dependently from each set of titrations and is well defined;
exactly the same value, 0.369 0.003, is found for ribo-
somes and subunits at 5 mM and 10 mM magnesium, falling
to 0.352 at 20 mM. Since the anisotropy cannot exceed 0.400,
this value reflects a firm binding of the small molecule to the
ribosome, with little segmental mobility. The use of different
values for different sites in the computer fit (see Materials and
methods) was unnecessary.
A second measure of the degree of binding is provided by
the relative change in intensity when the free fluorophore
becomes bound. This is independent of the anisotropy values.
However, the intensity data proved less reproducible than the
anisotropy data (the intensity factors, defined below, had an
error of typically 10-20%o), perhaps owing to small adsorp-
tion effects or titration errors. The intensity increase was
therefore not used to evaluate the degree of binding; although
the ratio of intensities of bound and free demeclocycline is re-
quired for exact evaluation of the anisotropy (see Materials
and methods), this does not need to be so accurately known.
Reversibility of demeclocycline binding
In the measurement of equilibrium constants it is important
to ascertain that a dynamic equilibrium is reached. This was
tested by dilution experiments: anisotropy in a diluted sample
122
Second (weak) binding Reference
n, K2 (M- 1) n2
0.72 5.7 x 102 509 a
0.6-1 - - b
0.4-0.6 0.2-1.2 x 107 0.9 c
_ 7.8 x 103 - d
was not higher than in a sample in which ribosomes and
demeclocycline were mixed at a corresponding, low concen-
tration. Moreover, titrations in both directions (ribosomes to
demeclocycline or vice versa) gave the same results.
Interpretation of binding data
Anisotropy was measured for increasing concentrations of
demeclocycline added to ribosomal particles at various initial
concentrations. These are shown for 5 mM magnesium in
Figure 2. Qualitatively, it is clear that: (i) for any given
demeclocycline concentration, the observed anisotropy is
greater for a higher ribosome concentration, since a greater
fraction of the antibiotic is bound; (ii) conversely, for any
given ribosome concentration, the observed, average aniso-
tropy decreases as more demeclocycline is added, (iii) the
ability of 50S subunits to immobilise demeclocycline is con-
spicuously less than that of 30S subunits or 70S ribosomes.
The titration curves were evaluated quantitatively by least-
squares refinement by applying the theoretical binding equa-
tions for five different models of the number and kind of
binding site for demeclocycline on the ribosome: (a) one bin-
ding site '1', with a binding constant K1; (b) two independent
binding sites 'la' and '1 b', with respective binding constants
Kla and Klb; (c) a large, unspecified number n2 of binding
sites '2', with equal binding constants K2; (d) one binding site
'1,, with a characteristic binding constant K1, and a large,
unspecified number n2 of sites '2', with binding constants K2
that are equal to each other but not necessarily to K1; (e) two
independent binding sites 'la' and 'lb', with different bin-
ding constants Kla and Klb, and a large, unspecified number
n2 of sites '2', with binding constants K2 equal to each other
but different from Kla and Klb.
The resulting optimum fits for 30S subunits with models
(a) and (c) are shown in Figure 3a and b. For these models,
the fit is not satisfactory, whereas it is acceptable for model
(d), as shown in Figure 2a. For numerical assessment, the
normalised sums of residuals r were compared; r-values for
all models, together with optimised binding parameters, are
given in Table II. Comparison of the models by inspection of
the fit or by the r-value criterion gives the following results.
(i) No agreement between observed and calculated aniso-
tropies can be reached, if only one [model (a)] or two [model
(b)] binding sites are assumed. The best-fit theoretical binding
shows a curvature which is systematically too high (e.g.,
Figure 3a). This applies to all ribosomal particles.
(ii) If a large number of similar binding sites is assumed
[model (c)], corresponding to multiple unspecific binding of
the antibiotic, the data from 50S subunits can be matched
 Tetracycline binding to nbosomes

0.35 D 0.35 _
a G
-
**

0.25 r- * *
0.25 **5
*

\*\
*
0 *

0.15 *
* ~ - * **
*
0.15-~~ ~ ~
~~~~~~~~~~~
I-* 0.15
I -1
0.35~ 0.35
b b
0- *
0
L-- 0.25 L CL
0
0 0.25 * *
0 LA

~~ ~ *
< 0.5 * -* * *--
*
- * * * -
0.15 * *** _ 0.15 * *
1- I
0.35 0.35 -

**~ C C I
_ * T~~~~~

0.25* _ _ _ 0.25 -
---* -* *

0.15
0 200 400 600
Demeclocycline Qdded (pmol)
Fig. 2. Fluorescence emission anisotropy of demeclocycline in the presence
of ribosomal particles at 5 mM magnesium. (a) 30S subunits (bottom to
top): 50, 75, 160 and 420 pmol. (b) 50S subunits: 76 and 190 pmol. (c) 70S
ribosomes: 75, 150 and 312 pmol. The sample volume was - 300 Il. The
curves are the calculated best-fit curves for the appropriate binding models
(see text).
very well [e.g., Figure 2; contrast model (a), Figure 3c]. As
expected for weak binding, the number of binding sites and
the binding constant cannot be determined independently of
one another; equally good fits are obtained for all n > 10.
However, the product of n2 and K2 is well defined. If model
(d) is applied to the 50S subunits then the fit is still good but
K1 has a low value, showing that the single binding site is
redundant. If model (c) is applied to the 70S or 30S particles
then the fit is poor and the theoretical binding shows a cur-
vature which is systematically too low (e.g., Figure 3b).
(iii) Adequate fitting of the binding behaviour of 70S and 30S
particles is not possible unless strong binding together with
multiple weak interactions is assumed [models (d) and (e)].
Calculations based on one strong binding site (n1 = 1) and
many of a second type (n2 >5) allow good fitting of the
observed 70S and 30S data. Moreover, if the more complex
model (e), with nia = n1b = 1 and n2 large, is applied, then it
simplifies during refinement to model (d) in that either Kia or
Kib converges to a low value, insignificantly different from
0.1 5 - =* -* -* *
!
0 200 400 600
Demeclocycline added (pmol)
Fig. 3. Some examples of binding models rejected on the basis of inade-
quately good fit to the data. (a) 30S subunits, one binding site; points as in
Figure 2a. (b) 30S subunits, many equivalent binding sites; points as in
Figure 2a. (c) 50S subunits, one binding site; points as in Figure 2b. For
quantitative comparison, see text and Table II.
zero.
In summary, the binding data point unambiguously to
model (c) for 50S subunits and to model (d) for 70S and 30S
particles.
Magnesium dependence of equilibrium constants
For 70S ribosomes, additional data were collected at 10 mM
and 20 mM magnesium. The qualitative pattern of demeclo-
cycline binding and the numbers of binding sites at these con-
centrations were the same as those at 5 mM, but the
equilibrium constants for strong and for weak binding are
somewhat reduced at the higher magnesium concentrations.
The equilibrium constants obtained from the relevant binding
models for all particles and magnesium concentrations are
summarised in Table III.
Sources of error in equilibrium constants
The calculated standard errors of the parameters obtained in
the refinement procedure were low; most values lay between
10o and 50%o and none exceeded this limit. However, various
sources of systematic error can influence the absolute values
123
B. Epe and P. Woolley

Table II. Comparison of optimum fits for various binding models (5 mM Mg; association constants in (JuM) 1, residual function r defined in Materials and
methods
Model Figure K (n = 1) Klb (n = 1) K2 (n= 10) 104 r
30S One strong site 3a 3.8 --- 592
Two strong sites - 1.3 1.2 --- 70
Many weak sites 3b --- --- 0.10 531
One strong site
plus weak sites 2a 2.2 --- 0.029 27
Two strong sites
plus weak sites - 2.1 0.19 0.018 26

50S One strong site 3c 0.70 --- --- 509

Two strong sites - 0.30 0.30 --- 249
Many weak sites 2b --- --- 0.035 30
One strong site
plus weak sites - 0.041 --- 0.032 31
70S One strong site - 9.1 --- --- 951
Two strong sites - 2.3 2.5 --- 243
Many weak sites --- --- --- 0.20 317
One strong site
plus weak sites 2c 3.2 - 0.082 73
Two strong sites
plus weak sites - 3.3 0.042 0.078 74

Table HI. Summary of association constants for tetracycline with 70S However, the qualitative conclusions about the pattern of the
ribosomes and with 30S and 50S subunits binding, the numbers of binding sites and the trend towards
weaker binding at higher magnesium concentration are unaf-
Particle Mg concen- Strong binding Weak binding (n2 2 10) fected.
tration K1 n2 K2
(mM) (M- l) (M - l) Discussion
30S 5 2.2 x 106 0.29x 106 The results of this study strongly support the description of
50S 5 - 0.35 x 106 tetracycline binding involving a single, strong binding site and
70S 5 3.2 x 106 0.82x 106 a large number of much weaker sites. The anisotropy method
70S 10 2.0 x 106 0.68x 106 allows a determination which, as discussed above, overcomes
70S 20 1.1 x 106 0.40 x106
many of the experimental problems associated with determin-
ing the degree of binding. A weakness of the method is the
need for non-linear least-squares curve-fitting, but, as shown
above, it was possible to keep the number of parameters
of the equilibrium constants. (i) Small deviations from the 1:1 reasonably small. Quantitatively, our results are in best agree-
stoichiometry assumed for the strong interactions caused, - ment with those of Streltsov et al. (1974, 1975) (cf. Tables I,
for example, by the presence of partially inactive ribosomes III); the discrepancies between these and other results, such as
- would result in an artificially low value for the constant the variation in K1 by 3-4 orders of magnitude, cannot light-
K1. (ii) As mentioned above, the greatest single source of er- ly be explained away, but we believe that the anisotropy
ror lies in the intensity ratios for bound vis-a-vis free demeclo- method is particularly suited to the determination of
cycline. An inaccuracy in these ratios would affect the ab- demeclocycline binding, while its reliability is confirmed by
solute values of the constants not, however, the observed
- the conspicuous difference between the good (Figure 2) and
pattern of binding. (iii) For 30S and 50S particles, the the bad (Figure 3) curve-fits over a wide range of concentra-
assumption of a large number of arbitrarily weak sites of type tions of demeclocycline and of ribosomal particles.
2 (n2 > 10) cannot be falsified by the experimental data, since In this study we have for the first time attempted a quanti-
the assumption of a smaller number of weak binding sites (n2 tative comparision of the binding of a tetracycline by 70S,
= 4-10) would not impair significantly the overall fit and 30S and 50S particles; the conclusion that the strong binding
would be compensated for by a lower value of K1. However, site for demeclocycline is associated primarily with the 30S
the unambiguous result for 50S subunits suggests that this subunit seems unambiguous. The fact that the strong binding
assumption is correct for the other ribosomal particles as appears to be significantly (50%) stronger on the 70S particle
well. suggests that the binding site on the 30S subunit is located
By appropriate adjustment of parameter values it was sufficiently close to the 50S subunit to be influenced by it,
shown that each of the three sources of systematic error could even though no strong interaction occurs with the larger sub-
be responsible for an error in K1 and/or in n2K2 of up to 10%. unit alone. Predominant binding to the 30S subunit is consis-
124

tent with the pattern of photo-affinity labelling of tetracycline
to ribosomal proteins (reviewed by Cooperman, 1980;
Goldman et al., 1980, 1983), in which the principal labelled
product is protein S7. In addition, a tetracycline-resistant mu-
tant of Bacillus subtilis has been found to contain a modified
protein S10 (Williams and Smith, 1979), although the
mechanistic relevance of this finding is not yet clear (Gale et
al., 1981).
On the assumption that the strong binding site is responsi-
ble for the effect of tetracycline upon tRNA binding, the
equilibrium constants determined in this work can be combin-
ed with reported constants (e.g., Holschuh et al., 1981) for
tRNA binding to the A site in order to calculate the concen-
tration at which tetracyclines can displace tRNA. For ribo-
somes and tRNA concentrations of 10-6 M notable in-
terference would occur at antibiotic concentrations around
10-4 M. This is in agreement with the finding (reviewed by
Brown and Ireland, 1978) that the selective anti-microbial ac-
tivity of tetracyclines arises from the suicidal propensity of
bacteria to accumulate the antibiotics by active transport,
since the external concentration for bactericidal activity is on-
ly a few micromolar in vivo (Forth et al., 1977) and in vitro
(Fussganger, 1958). However, it does not presuppose a par-
ticular mechanism (e.g., direct blocking or allosteric weaken-
ing of the A site) for the inhibitory action of tetracycline.
All three ribosomal particles show a large number of addi-
tional binding sites which can be regarded as unspecific. The
weak binding capacity increases with the size of the particle
(30S < 50S < 70S) and is, as expected, approximately ad-
ditive. The fact that n2K2 for 70S is somewhat greater than for
30S and 50S combined may, if significant, perhaps be at-
tributed to the formation of unspecific binding sites in the
crevice between the subunits. It cannot at present be excluded
that the weak binding sites have a function: even for a large
molar excess of ribosomes over antibiotic the weak sites,
because of their number, account for 30- 40%0 of the overall
binding.
In our study, the effect of an increase in magnesium con-
centration was to reduce the affinity of both strong and weak
tetracycline binding by the ribosome. This is in agreement
with White and Cantor (1971). It also suggests - but does
not prove - that the tetracycline may be binding to RNA.
There is some disagreement in the literature over the magnes-
ium dependence of inhibition of ribosomal polypeptide syn-
thesis by tetracycline: while Bodley and Zieve (1969) found no
inhibition of Phe-tRNA binding below 10 mM magnesium
and strong inhibition at all concentrations above this, Tritton
(1977) found inhibition of poly(Phe) synthesis only below
20 mM magnesium. Since a high magnesium concentration
generally strengthens RNA-RNA interactions and, according
to our data, seems to weaken tetracycline-ribosome inter-
actions, we could expect the primary effect of a high mag-
nesium concentration to be a reduction in the effectiveness of
tetracycline as an inhibitor of the binding of tRNA by the
ribosome, in agreement with the result of Tritton. However,
the exact mechanism of tetracycline action is not known; we
shall discuss elsewhere the question of competitive binding
between tetracycline and tRNA.
Materials and methods
6-Demethylchlortetracycline (Ledermycin' demeclocycline) was kindly
donated by Cyanamid GmbH (Wolfratshausen, FRG). Ribosomes and ribo-
somal subunits were prepared according to Hapke and Noll (1976) and Dijk
and Littlechild (1979).
Tetracycline binding to ribosomes
Fluorimetric measurements were performed at 25°C in a 5 x 5 mm quartz
cell. The buffer was: NH4CI 50 mM, Mg acetate, 5, 10 or 20 mM as in-
dicated, dithioerythritol 0.1 mM, Tris.HCI 20 mM, pH 7.5 (cf. Luhrmann,
1980). In most cases, increasing amounts (80-600 pmol) of stock solutions of
demeclocycline (5 mg/l; 10 AM) were added to a given concentration (between
0.1 and I AM) of ribosomal particles in 250 Al buffer in the optical cell. Im-
mediately before titration, the ribosomes or subunits were incubated in the
measurement cell at 37°C for 5 min. After each addition of demeclocycline
the solution was allowed to equilibrate for 15 min at 25°C.
Fluorescence measurements were made in an SLM 8000 DS spectrofluori-
meter with digital data acquisition (for details see Steinhauser et al., 1982).
This apparatus has a 450 W Xe lamp as light source and a double-grating ex-
citation and single-grating emission monochromator. Polarised emission spec-
tra were taken with vertically polarised excitation at 380 nm; the emission
range was 500-540 nm; excitation and emission bandwidths were 8 nm.
Exact background correction was applied by conducting simultaneous,
parallel titrations with omission of demeclocycline. For each point the
anisotropy and the anisotropy-corrected total intensity were calculated; these
were then averaged over the measured emission range. Each data-point was
measured at least four times and the average value was used in the calculation
(below).
Curve-fitting was carried out on a Hewlett-Packard 9825 B calculator with
23 kbyte store. The multi-parameter, non-linear least-squares programme was
adapted from a routine, originally written by G.M. Sheldrick at Cambridge
University, which allows the simultaneous refinement of several parameters.
To carry out the curve-fitting, the binding equations were first formulated:
Ri + T Kj Ri.T (i = la,lb,2)
where Rj is a binding site of type i (cf. models (a) -(e), above), T is a
demeclocycline molecule and K, the associated binding constant. If the total
concentration of ribosomes is [R]o and that of demeclocycline [TIO, then
ni [R]o = [R]i + [Rj.-7 (i la,lb,2) =
and
[T7 [R. 7]
[ITo = +
where ni is therespective number of binding sites per ribosome.
Substitution of the [Ri. 71 terms following from the mass-action equation
above and assumption of the numerical values for the parameters of binding
Ki and ni lead to four non-linear simultaneousofequations in which the only
unknowns are Rua, Rlb, R2 and T. Elimination the Rj leads to the single ex-
pression for T:
[1 = [71 (1 + [Ro] Ki
-niI +Kj[71
which is then solved for T by iteration, after which the other unknowns are
easily calculated.
When the concentrations are known, the overall anisotropy <a> of the
solution is calculated from the anisotropies aj of the single species present by
Weber's law:
IV,
aj fj cj
<a> =
Ef cj
where for each fluorescing species j the concentration is c. and the
fluorescence intensity per unit concentration is f. Since the scale off; is arbi-
trary, we definef for free demeclocycline to be 1.00, so all otherf values are
equal to the ratio of fluorescence intensities in bound and free states. is fA
therefore also called the 'intensity factor'.
Thus, with the assumption of values for the parameters Ki, ni, and aj, thefj
overall anisotropy <a> can be calculated, as a function of [R]O and [ on- 1O
then adjusted so
[T1O
ly, for all the [RIo and values in a set of titrations
as to optimise the fit
(e.g., in Figure 2a). The
of calculated and observ-
parameters are
ed < a > -values by the least-squares criterion. Of course, not all the
parameters needed to be refined: for example, the anisotropy of free
demeclocycline was measured independently andforfixed in the refinement; no
difference was found between anisotropy values demeclocycline bound to
strong and to weak sites, so these parameters were constrained to be identical;
the intensity factor for site lb is indeterminate, since site lb does not exist, so
the constraint fib fa was imposed; intensity factors were determined in-
=
dependently as far as possible and fixed in the refinement; since n2 proved in-
determinate, its value was fixed (e.g., n2 10) sowereas to determine
=
n2K2 by
kept at fixed values of
refinement of K2; for final evaluation, nla and nib
1 or 0. Since in most cases not all sites were included (some n 0) the
number of parameters refined was usually only two to four.
125
 B. Epe and P. Woolley

Quantitative comparison of the quality of fit is given by the r-factor, defin-

ed by:
_E(<a>obs - <a>Calc2
( <a>ob,)2
in which summation is over all data-points.

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Received on 19 September 1983; revised on 17 October 1983

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