Search PubMed⌕ Search

Biomedical subjects

R A Garrett

Publications and source records attributed to R A Garrett.

At least 127 records · Page 7Linked to original sources
PubMed1977

Physical studies on the conformation of ribosomal protein S4 from Escherichia coli.

Proton magnetic resonance, circular dichroism and infrared spectroscopy were used to investigate the secondary and tertiary structure of the 16-S RNA binding protein S4 from Escherichia coli ribosomes. The proton magnetic resonance spectra of protein S4 in ribosomal reconstitution and low-salt buffers were identical and showed little dipolar broadening of the peaks, suggesting that the protein had an open extended structure. A ring-current-shifted apolar methyl resonance in the high-field region of the spectrum, together with a perturbation of the tyrosine ring proton resonance in the low-field region, indicated the existence of a specific tertiary fold in the polypeptide chain. This structure disappeared on lowering the pH below 5 or on heating above 30 degrees C, both processes being reversible. Circular dichroism measurements on protein S4 showed an alpha-helix content of 32% in reconstitution buffer compared with 26% in low-salt buffer. Heating the protein solution in reconstitution buffer above 35 degrees C reversibly disrupted this extra helix. Infrared studies on both solid films and solutions of protein S4 indicated the presence of little or no beta-structure. These results correlate well with the known RNA binding properties of protein S4.

Circular Dichroism↗
PubMed1977

A trypsin-resistant fragment from complexes of ribosomal protein S4 with 16-S RNA of Escherichia coli and from the uncomplexed protein.

A fragment of ribosomal protein S4 was prepared by limited trypsin degestion of a specific complex between protein S4 and 16-S RNA. It was characterised for amino acid sequence and the N-terminal 46 amino acids were found to be absent. An intermediate fragment, cut at Arg-43, was also observed at low trypsin concentrations. Evidence is presented that the protected fragment constitutes the primary RNA-binding region of the protein. No smaller protein fragments were found that rebound to the RNA. A mechanism for the degradation of the N-terminal region of the protein is proposed and two probable functions of the excised region are given. Under milder trypsin digestion conditions than for the complex, the same fragment, cut at Arg-46, was also prepared from the free protein. This result, together with that from a control experiment, indicates that at least within this local region, the protein conformation is conserved in both the free protein and the protein-RNA complex. This is the first direct evidence for the conservation of conformation in a protein when both complexed and uncomplexed with a ribosomal RNA.

Amino Acid Sequence↗
PubMed1977

Accessibility of proteins in 50S ribosomal subunits of Escherichia coli to antibodies: an ultracentrifugation study.

The accessibility of each of the proteins on the 50S ribosomal subunit of Escherichia coli was investigated by establishing whether immunoglobulins (IgG), specific for each of the 34 proteins from the 50S subunit, were able to bind to the 50S subunit. The main criterion for accessibility was the formation of specific antibody-50S subunit complexes that could be detected by means of analytical ultracentrifugation. The proteins fell into two main groups. Immunoglobulins against proteins L1, L2, L3, L4, L5, L6, L7/L12, L8, L9, L10, L11, L14, L15, L16, L17, L18, L19, L20, L21, L22, L23, L25, L26, L27 and L30 gave large amounts of complex (20-100%) and, therefore, these proteins were considered to be accessible on the surface of the 50S ribosomal subunit. The antibodies against the remaining proteins L13, L24, L28, L29 and L31 to L34 produced small amounts of complexes (10-20%). Since their effects were unequivocably stronger than those obtained with IgG's from sera of non-immunized animals, the results indicate that these proteins are probably also accessible. Nonetheless, from the ultracentrifugation studies alone definite conclusions about the exposure of the latter group of proteins could not be drawn.

Antibody Specificity↗
PubMed1977

An RNA core in the 30S ribosomal subunit of Escherichia coli and its structural and functional significance.

1. Evidence is presented for the occurrence of a very stable RNA core (S4-RNA) in "native" 16S RNA that is also present in the 30S subunit of Escherichia coli. A model giving the approximate location of this RNA core in the 30S subunit is presented. 2. It is proposed (a) that this S4-RNA acts as a nucleus for the assembly of the 30S subunit, and (b) that a small class of "linkage" proteins, including S4, further facilitate the assembly of the proteins to the RNA, thereby explaining some of the "cooperative" effects that are observed during in vitro assembly. 3. Evidence for the importance of the RNA core in the functioning of the ribosome is discussed.

Bacteria↗
PubMed1977

Alteration of 5S RNA conformation by ribosomal proteins L18 and L25.

The effects of ribosomal proteins L18, L25 and L5 on the conformation of 5S RNA have been studied by circular dichroism and temperature dependent ultraviolet absorbance. The circular dichroism spectrum of native 5S RNA is characterized in the near ultraviolet by a large positive band at 267 nm and a small negative band at 298 nm. The greatest perturbation in the spectrum was produced by protein L18 which induced a 20% increase in the 267 nm band and no change in the 298 nm band. By contrast, protein L25 caused a small decrease in both bands. No effect was observed with protein L5. Simultaneous binding of proteins L18 and L25 resulted in CD changes equivalent to the sum of their independent effects. The UV absorbance thermal denaturation profile of the 5S RNA L18 complex lacked the pre-melting behavior characteristic of 5S RNA. Protein L25 had no effect on the 5S RNA melting profile. We concluded that protein L18 increases the secondary, and possible the tertiary structure of 5S RNA, and exerts a minor stabilizing effect on its conformation while protein L25 causes a small decrease in 5S RNA secondary structure. The implications of these findings for ribosome assembly and function are discussed.

Chemical Phenomena↗
PubMed1976

The binding site of protein L1 ON 23-S ribosomal RNA of Escherichia coli. 2. Identification of the rna region contained in the L1 ribonucleoproteins and determination of the order of the RNA subfragments within this region.

Ribonucleoproteins were obtained by T1 ribonuclease digestion of reconstitued complexes of ribosomal protein L1 AND 23-S RNA from Escherichia coli. The RNA region of the main ribonucleoprotein 2 was totally digested with T1 ribonuclease. The oligonucleotide products were characterised and they showed that this region comprises 148 nucleotides located between the 550th and 1000th necleotides from the 3' end of the 23-S RNA. Of the other two ribonucleoproteins, the largest ribonucleoprotein 1 contained an extra RNA sequence, of at least 15 nucleotides, that was located at the 5' end of the RNA region. The smallest ribonucleoprotein 3 lacked an RNA section towards the 3' end of the region. The order of the RNA subfragments and the enzymic cutting positions in the whole RNA region are given for the ribonucleoproteins. It is shown that protein L1 most strongly protects a continuous section of 115 nucleotides at the 5' end of the main RNA region. Finally, evidence is presented for a methylated base, and for two sequence heterogeneities, in this region of the 23-S RNA.

Alkaline Phosphatase↗
PubMed1976

Molecular model for 5-S RNA. A small-angle x-ray scattering study of native, denatured and aggregated 5-S RNA from Escherichia coli ribosomes.

A tertiary structural model is suggested for Escherichia coli 5-S RNA that consists of one large and two small double helices arranged in the form of the letter Y. This model is consistent with the small-angle X-ray scattering data of native 5-S RNA, measured in the angular range 20 less than or equal to 140 mrad. The radium of gyration is 3.61 +- 0.1 Nm. Denatured 5-S RNA yields a much lower radius of gyration, 2.7 nm, which might indicate that during denaturation one minor double-helical arm of the Y-shaped structure partially collapses into single-stranded areas. At high concentrations (60 mg/ml) of 5-S RNA, the X-ray scattering data indicate that 5-S RNA is aggregated.

Base Sequence↗
PubMed1976

An investigation of the binding sites of proteins S8, L23 and L24 on the ribosomal RNAs of Escherichia coli by electron microscopy.

An electron microscopic method was used to investigate the binding regions of proteins S8 on 16S RNA, and proteins L23 and L24 on 23S RNA. Regions of the RNA that were not stabilised by the protein were completely denatured in 80% dimethyl-sulfoxide. The lengths of these denatured RNA regions were compared with that of the whole denatured RNA. Conclusions are drawn concerning the approximate location of the three proteins and these results are correlated with both RNA structural data and RNA sequence data on the RNA binding regions of the proteins.

Bacterial Proteins↗
PubMed1975

Location and characteristics of ribosomal protein binding sites in the 16S RNA of Escherichia coli.

Specific binding sites for five proteins of the Escherichia coli 30S ribosomal subunit have been located within the 16S RNA. The sites are structurally diverse and range in size from 40 to 500 nucleotides; their functional integrity appears to depend upon both the secondary structure and conformation of the RNA molecule. Evidence is presented which indicates that additional proteins interact with the RNA at later stages of subunit assembly.

Bacterial Proteins↗