Prediction of the DNA-recognizing supersecondary protein structure, alpha helix-turn-alpha helix, using the modified Ohlendorf-Anderson-Matthews method of necessary stereochemical requirements

A method for prediction of DNA-recognizing supersecondary structure alpha-helix--turn--alpha-helix localization in an amino acid sequence of any protein is described. The method allows to predict this structure in segment 67-89 of E. coli ribosomal protein L7/L12 and in corresponding segments of L7/L12 analogues from other six bacteria and spinach chloroplasts.     

Location of the 5.8S rRNA gene of Saccharomyces cerevisiae.

Direct DNA sequence analysis of Saccharomyces cerevisiae ribosomal DNA cloned in an Escherichia coli plasmid revealed part of the structural gene for 5.8S rRNA at one end of a 700-base-pair EcoRI fragment. Taken with the previously established EcoRI restriction map of the ribosomal repeat unit, this sequence establishes that the yeast 5.8S RNA segment is located between the 18S and 28S segments in the 42S rRNA precursor and in the DNA which codes for it.     

Sequence of the chloroplast 16S rRNA gene and its surrounding regions of Chlamydomonas reinhardii.

The sequence of a 2 kb DNA fragment containing the chloroplast 16S ribosomal RNA gene from Chlamydomonas reinhardii and its flanking regions has been determined. The algal 16S rRNA sequence (1475 nucleotides) and secondary structure are highly related to those found in bacteria and in the chloroplasts of higher plants. In contrast, the flanking regions are very different. In C. reinhardii the 16S rRNA gene is surrounded by AT rich segments of about 180 bases, which are followed by a long stretch of complementary bases separated from each other by 1833 nucleotides. It is likely that these structures play an important role in the folding and processing of the precursor of 16S rRNA. The primary and secondary structures of the binding sites of two ribosomal proteins in the 16SrRNAs of E. coli and C. reinhardii are considerably related.     

Structure of a Two-Domain N-Terminal Fragment of Ribosomal Protein L10 from Methanococcus jannaschii Reveals a Specific Piece of the Archaeal Ribosomal Stalk

Ribosomal stalk is involved in the formation of the so-called “GTPase-associated site” and plays a key role in the interaction of ribosome with translation factors and in the control of translation accuracy. The stalk is formed by two or three copies of the L7/L12 dimer bound to the C-terminal tail of protein L10. The N-terminal domain of L10 binds to a segment of domain II of 23S rRNA near the binding site for ribosomal protein L11. The structure of bacterial L10 in complex with three L7/L12 N-terminal dimers has been determined in the isolated state, and the structure of the first third of archaeal L10 bound to domain II of 23S rRNA has been solved within the Haloarcula marismortui 50S ribosomal subunit. A close structural similarity between the RNA-binding domain of archaeal L10 and the RNA-binding domain of bacterial L10 has been demonstrated. In this work, a long RNA-binding N-terminal fragment of L10 from Methanococcus jannaschii has been isolated and crystallized. The crystal structure of this fragment (which encompasses two-thirds of the protein) has been solved at 1.6 Å resolution. The model presented shows the structure of the RNA-binding domain and the structure of the adjacent domain that exist in archaeal L10 and eukaryotic P0 proteins only. Furthermore, our model incorporated into the structure of the H. marismortui 50S ribosomal subunit allows clarification of the structure of the archaeal ribosomal stalk base.     

The genes for the ribosomal proteins S12 and S7 are clustered with the gene for the EF-Tu protein on the chloroplast genome of Euglena gracilis.

We characterize a DNA segment of the Euglena gracilis chloroplast DNA fragment Eco . N by nucleotide sequencing and S1 nuclease analysis. We show that this region, which is upstream of the previously sequenced tuf A gene, contains the genes for the ribosomal proteins S12 and S7. The gene arrangement is 5'-rps 12-80 bp spacer-rps 7-174 bp spacer-tuf A, somewhat similar to the str operon of E. coli. The chloroplast S12 and S7 proteins contain 124 and 155 aminoacids, respectively, and are to 68% and 38% homologous with the corresponding E. coli proteins. The region is transcribed into a distronic mRNA of about 1.1 to 1.2 kb. The rps 12 and rps 7 genes, contrary to the tuf A gene, are not split.     

N-terminal domain, residues 1-91, of ribosomal protein TL5 from Thermus thermophilus binds specifically and strongly to the region of 5S rRNA containing loop E.

In this work we show for the first time that the overproduced N-terminal fragment (residues 1-91) of ribosomal protein TL5 binds specifically to 5S rRNA and that the region of this fragment containing residues 80-91 is a necessity for its RNA-binding activity. The fragment of Escherichia coli 5S rRNA protected by TL5 against RNase A hydrolysis was isolated and sequenced. This 39 nucleotides fragment contains loop E and helices IV and V of 5S rRNA. The isolated RNA fragment forms stable complexes with TL5 and its N-terminal domain. Crystals of TL5 in complex with the RNA fragment diffracting to 2.75 A resolution were obtained.     

Proteolysis of ribosomal protein S1 from <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Thermus thermophilus</Emphasis> leads to formation of two different fragments

As a result of limited tryptic proteolysis of S1 ribosomal protein (molecular mass 60 kD) from Thermus thermophilus, 25 N-terminal amino acid residues and 71 C-terminal amino acid residues are split off and a stable high-molecular-weight fragment with molecular mass of 49 kD is formed that retains RNA-binding properties and is capable of interacting with 30S ribosomal subunit. Earlier, application of a similar procedure for the formation of a fragment of S1 protein from Escherichia coli resulted in splitting of 171 N-terminal amino acid residues with the formation of a 41.3 kD fragment that possesses RNA-binding properties only. Thus, in spite of high homology between E. coli and T. thermophilus proteins, the proteolysis leads to the formation of two different fragments, which points, in our opinion, to the fact of significant differences between their structures.     

Isolation and physical study of the 13S fragment of 16S RNA and its complex with ribosomal protein S4

A fragment of E. coli 16S RNA has been obtained by its hydrolysis with pancreatic RNAase A coupled to Sepharose 4B. This fragment has a molecular weight of 170 000 and a sedimentation coefficient of 13S. It does not aggregate in solution and binds with the ribosomal protein S4. The 13S fragment and it complex with the protein S4 have been studied by different physical methods in the first place, by neutron scattering. It has been shown that this fragment is compact in solution. The radii of gyration of the fragment (50 +/- 3 A) and of the protein S4 within the complex (17 +/- 3 A) coincide, within limits of experimental error, with the radii of gyration for the free RNA fragment (47 +/- 2 A) and the free ribosomal protein S4 in solution (18 +/- 2 A). Hence, the conclusion is made that the compactness of the 13S fragment of the 16S RNA and the ribosomal protein S4 does not change at the complex formation. The compact 13S fragment of the 16S RNA is shown to be contrast matched in the H2O/D2O mixture containing 70% D2O which corresponds to its partial specific volume v equal to 0.537 cm3/g.     

Maize chloroplast DNA encodes a protein sequence homologous to the bacterial ribosome assembly protein S4.

A cloned restriction fragment of maize chloroplast DNA (Bam H1 fragment 5) is shown to contain an open reading frame which encodes a basic protein of 201 amino acid residues with 40-50% sequence homology to E. coli ribosomal protein S4. Based on the experimentally determined sequence homology between the highly conserved bacterial ribosomal protein L12 and its chloroplast homologue (Bartsch M., Kimura, M. and Subramanian, A.R. (1982) Proc. Natl. Acad. Sci. USA 79, 6871), we conclude that this reading frame represents the maize chloroplast S4 gene. The nucleotide sequence of a 1100 base pair DNA segment containing the putative gene is presented.     

Isolation of Euglena gracilis chloroplast 5S ribosomal RNA and mapping the 5S rRNA gene on chloroplast DNA.

Ribosomal RNA (5S) from Euglena gracilis chloroplasts was isolated by preparative electrophoresis, labeled in vitro with 125I, and hybridized to restriction nuclease fragments from chloroplast DNA or cloned chloroplast DNA segments. Euglena chloroplast 5S rRNA is encoded in the chloroplast genome. The coding region of 5S rRNA has been positioned within the 5.6 kilobase pair (kbp) repeat which also codes for 16S and 23S rRNA. There are three 5S rRNA genes on the 130-kbp genome. The order of RNAs within a single repeat is 16S-23S-5S. The organization and size of the Euglena chloroplast ribosomal repeat is very similar to the ribosomal RNA operons of Escherichia coli.     

Nucleotide sequence and linkage map position of the secX gene in maize chloroplast and evidence that it encodes a protein belonging to the 50S ribosomal subunit

The nucleotide sequence of the segment of maize chloroplast DNA lying between the map coordinate positions 32.59 and 32.98 Kb and containing the secX gene has been determined. The derived amino acid sequence of maize chloroplast secX is 95%, 87% and 62% identical to the corresponding derived amino acid sequences from two plant chloroplasts and Escherichia coli, respectively. It is also 70% identical to the experimentally determined amino acid sequence of a protein isolated from Bacillus stearothermophilus ribosomes. Separation of the 50S ribosomal subunit proteins of E. coli by reversed phase HPLC gave a peak which contained pure secX protein, as determined by N-terminal amino acid sequencing. Spinach chloroplast 50S subunit proteins separated by HPLC also gave a peak corresponding to pure secX protein. From these results we conclude that the secX gene in E. coli and in plant chloroplasts encodes a small (37-38 amino acid residues) ribosomal protein belonging to the 50S subunit. The same conclusion has been reached recently by A. Wada with respect to E. coli secX. In agreement with Wada, we name the secX protein L36. Its chloroplast gene is designated rpL36.     

A rice (Oryza sativa L.) cDNA encodes a protein sequence homologous to the eukaryotic ribosomal 5S RNA-binding protein

A rice (Oryza sativa L.) cDNA clone coding for the cytoplasmic ribosomal protein L5, which associates with 5 S rRNA for ribosome assembly, was cloned and its nucleotide sequence was determined. The primary structure of rice L5, deduced from the nucleotide sequence, contains 294 amino acids and has intriguing features some of which are also conserved in other eucaryotic homologues. These include: four clusters of basic amino acids, one of which may serve as a nucleolar localization signal; three repeated amino acid sequences; the conservation of glycine residues. This protein was identified as the nuclear-encoded cytoplasmic ribosomal protein L5 of rice by sequence similarity to other eucaryotic ribosomal 5 S RNA-binding proteins of rat, chicken, Xenopus laevis, and Saccharomyces cerevisiae. Rice L5 shares 51 to 62% amino acid sequence identity with the homologues. A group of ribosomal proteins from archaebacteria including Methanococcus vanniellii L18 and Halobacterium cutirubrum L13, which are known to be associated with 5 S rRNA, also related to rice L5 and the other eucaryotic counterparts, suggesting an evolutionary relationship in these ribosomal 5 S RNA-binding proteins.     

A cyanogen bromide fragment of S4 that specifically rebinds 16S RNA.

Escherichia coli ribosomal protein S4 was subjected to cyanogen bromide cleavage and was found to generate a complete cleavage product capable of rebinding 16S rRNA. This fragment, consisting of residues 1-103, was found to bind with an apparent association constant of 11 microM-1. This fragment was used in place of S4 in an in vitro reconstitution experiment. Although the particles formed had a protein composition not significantly different from reconstituted 30S ribosomal subunits, their sedimentation behavior was more like that of particles reconstituted without S4. These results indicate to us that, although residues 104-203 of S4 are involved in the assembly of the 30S ribosome, they are not necessary for the binding of S4 to 16S RNA. Taken with previous results, the domain of S4 involved in specific binding of 16S RNA can be confined to residues 47-103.     

Demonstration of the GC-rich common arm in yeast ribosomal 5.8S RNA via 500-MHz proton nuclear magnetic resonance and Overhauser enhancements

In this paper we report the first 1H NMR study of the base-paired secondary structure of yeast 5.8S RNA. On the basis of a combination of homonuclear Overhauser enhancements and temperature dependence of the proton 500-MHz NMR spectrum, we are able to identify and assign eight of the nine base pairs in the most thermally stable helical arm: G116.C137-C117.G136-C118.G135- C119.G134-C120.G133-U121.G132- U122.A131-G123.C130. This arm contains an unusually temperature-stable (to 71 degrees C) segment of four consecutive G.C base pairs. This work constitutes the most direct evidence to date for the existence and base-pair sequence of the GC-rich helix, which is common to most currently popular secondary structural models for eukaryotic 5.8S ribosomal RNA.     

A study of the thermophilic ribosomal protein S7 binding to the truncated S12-S7 intercistronic region provides more insight into the mechanism of regulation of the str operon of E. coli(1).

A study of the ability of His6-tagged ribosomal protein S7 of Thermus thermophilus to interact with the truncated S12-S7 intercistronic region of str mRNA of Escherichia coli has been described. A minimal S7 binding mRNA fragment is a part of the composite hairpin, with the termination codon of the S12 cistron on one side and the initiation codon of the next S7 cistron on the other. It has a length in the range of 63-103 nucleotides. The 63 nucleotide mRNA fragment, which corresponds to a putative S7 binding site, binds very poorly with S7. Tight RNA structure models, which behave as integral systems and link the S7 binding site with the translational regulation region of the hairpin, are suggested. This observation provides more insight into the mechanism of S7-directed autogenous control of translational coupling of str mRNA.     

A purified nucleoprotein fragment of the 30 S ribosomal subunit of Escherichia coli.

A '13 S' nucleoprotein fragment was isolated from a nuclease digest of Escherichia coli 30-S ribosomal subunits and purified to gel electrophoretic homogeneity. It contained two polynucleotides, of about 1.1 . 10(5) and 2.5 . 10(4) daltons, which separated when the fragment was deproteinized. The major protein components were S4, S7 and S9/11, with S15, S16, S18, S19 and S20 present in reduced amount.     

Localisation of part of the binding sites of 30S ribosomal proteins S4 and S20 in a small uninterrupted fragment of 16S RNA.

Analyses of the T1 ribonuclease-alkaline phosphatase fingerprint of a continuous fragment of the 16S rRNA, 170-230 nucleotides long, isolated from the products of autodigestion of 30S ribosome subunits show that it contains a sequence near the 5'-phosphate terminus of intact 16S rRNA and corresponds to segment H'-M of this molecule as defined by Ehresmann et al [29]. Incubation of this fragment with total 30S ribosomal proteins under reconstitution conditions leads to the formation of a complex containing proteins S4, S20, and one or both of proteins S16 and S17. The stoichiometry of these proteins in the complex is discussed.