Download Conservation of Primary Structure in Bacterial Ribosomal Protein

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861
released from the interchain bonds by the reducing agent were unable to form disulphide
bridges and the chains remained principally monomeric as judged by polyacrylamide-gel
electrophoresis in 0.1 % sodium dodecyl sulphate.
When solutions of heavy or light chains were adjusted to pH7.5, disulphide bonds
were able to form and both heavy and light chains dimerized. Heavy chains formed
dimers far more rapidly than light chains.
Mixtures of heavy and light chains assembled into intact immunoglobulin molecules
(HzLz) by the H2 pathway
or the HL pathway
[H+L -+ HL; H L + H L
-+
HzL2 or H L + H
-+
HzL; HzL -+ H2L21
In the proteins studied, assembly occurred by both pathways, but the predominance of a
particular pathway varied with different immunoglobulins. Experiments with different
concentrations of heavy and light chains indicated that the HL pathway was favoured
when light chains were present in great excess.
When the light or heavy chains were replaced by the appropriate chains from different
proteins, hybrid molecules were formed at a rate similar to the autologous assembly
process, although the predominance of a particular pathway was sometimes altered.
Conservation of Primary Structure in Bacterial Ribosomal Protein
ALASTAIR T. MATHESON, MAKOTO YAGUCHI and LOUIS P. VISENTIN
Division of Biological Sciences, National Research Councilof Canada, Ottawa, Canada,
KlAOR6
Functional (Higo et al., 1973), immunological (Isono et al., 1973) and sequence data
(Yaguchi et al., 1973) on the ribosomal proteins from various bacterial sources suggest
considerable conservation of protein structure during the evolutionary development of
the bacterial ribosome. To determine the effects of evolutionary pressure, such as extremes in temperature and ionic environment, on the primary structure of bacterial
ribosomal proteins, we have purified the 21 proteins from the 3 0 s ribosomal subunit of
Escherichia coli, the thermophile Bacillus stearothermophilus and the extreme halophile
Halobacterium cutirubrum. The amino acid sequence of the first 30-40 residues was
determined using the Beckman sequencer model 89OC.
The N-terminal residues of the ribosomal proteins from both the thermophile and
halophile show the same non-random distribution of certain amino acids (alanine,
methionine and serine) as is found in mesophilic proteins (Waller, 1963). In all three
bacteria the major N-terminal residues in the 30s ribosomal proteins are alanine $
methionine>serine(e.g. in E. coli:46%alanine, 14%methionineand9xserine), whereas
in the 50s ribosomal proteins methionineaalanine>serine (e.g. in E. coli: 33 % methionine, 24% alanine and 10% serine). These results suggest conservation of specific Nterminal residues in the bacterial ribosomal proteins.
The amino acid sequence data obtained from the individual 3 0 s ribosomal proteins of
B. stearothermophilus show no evidence of sequence homologies among the 21 proteins
in the 30s subunit, at least not in the N-terminal region (the first 30-40residues). Similar
results have previously been obtained with the 3 0 s ribosomal proteins of E. coli(Yaguchi
et al., 1973; Wittmann-Liebold, 1973). However, when the amino acid sequences of
equivalent ribosomal proteins from E. coli and B. stearothermophilus are compared substantial regions of sequence homology are observed. In Table 1 the first 12 residues of
10 equivalent 3 0 s ribosomal proteins from E. coli and B. stearothermophilus are
compared.
When the amino acid compositions of the various positions are determined for the
total 3 0 s protein fraction (the sum of the individual proteins) some interesting observa-
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percentage of these are conserved in the equivalent B. stearothermophilus proteins. The
enrichment of the basic amino acids at the N-terminal portions of the 30s ribosomal
proteins is similar to the situation found in the histones.
These sequence data, in conjunction with the immunological (Isono et al., 1973) and
functional data (Higo et al., 1973), clearly indicate a large degree of conservation of
procaryotic ribosomal structure during evolution.
This paper is N.R.C.C. no. 14134.
Higo, K., Held, W., Kahan, L. & Nomura, M. (1973) Proc. Nat. Acud. Sci. US.70,944-948
Isono, K., Isono, S., Stoffler, G., Visentin, L. P., Yaguchi, M. & Matheson, A. T. (1973) Mol.
Gen. Genet. 127,191-195
Smithies, O., Gibson, D., Fanning, E. M., Goodfliesh, R. M., Gilman, J. G. & Ballantyne, D. L.
(1971) Biochemistry 10,4912-4921
Waller, J. P. (1963) J. Mol. Biol.7 , 483-496
Wittmann-Liebold, B. (1973) Hoppe-Seyler’s Z . Physiol. Chem. 354, 1415-1431
Yaguchi, M., Roy, C., Matheson, A. T. &Visentin, L. P. (1973) Cun.J. Biochem. 51,1215-1217
The Effect of Alkylammonium Ions on the Activity of Ribonucleic
Acid Polymerase
ALAN D. B. MALCOLM, G. JOAN MITCHELL and BOHDAN WASYLYK
Department of Biochemistry, University of Glasgow, Glasgow G12 8QQ, U.K.
It is not yet certain whether or not there is a local unwinding of the DNA duplex during
transcription by RNA polymerase (Burdon, 1973).
It has been shown that various small alkylammonium ions alter both the ‘melting’
point of DNA and the width of the ‘melting’curve. This latter responsearises from a difference in the effect of these ions on A-T and on G-C base pairs (Melchior 8~von
Hippel, 19731,and this is c o n k e d by studying the T,,,(‘melting temperature’) values of
different nucleic acids.
We have replaced K+ in the usual RNA polymerase assay (Burgess, 1969) with these
ions and have shown that there is a correlation between the resultant stimulation of
transcription and the lowering of the ‘melting’ point of the DNA template. Further, we
have shown, by varying the base composition of the DNA template, that there is also a
correlation between the stimulation of polymerase activity and the width of the ‘melting’
curves. It is not easy to account for these results except in terms of a model which involves
unwinding of the DNA during transcription. Thus, although the evidence is circumstantial, we feel that it strongly favours such a model.
Initiation and elongation of RNA synthesis are distinguishable steps in the mechanism of RNA polymerase, and we have shown that alkylammonium ions stimulate both
initiation and elongation (although to different extents) and that therefore an unwinding
of DNA is likely to be involved in both these steps.
When RNA synthesis is carried out at temperatures below 17°C there is a temporal
lag before production of RNA, and the length of this lag phaseincreases with lowering of
the temperature (Walter et al., 1967). It seems likely that this is a consequence of the need
to bring about an endothermic conformational change in the DNA template in order
that initiation may occur. This is presumably similar to a local ‘melting’ of the DNA and
in agreement with this we have shown that the presence of alkylammonium ions shortens
the length of this lag phase.
It is well known that the inhibitor of RNA synthesis, rifampicin, inhibits initiation
but not elongation. The effects of preincubation of template and enzyme in the presence
of alkylammonium ions on subsequent inhibition by rifampicin will also be described.
We thank Professor R. M. S. Smellie for his encouragement, John Greene for his assistance,
and the S.R.C.for financial support.
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