Structure and function of signal recognition particle RNA.
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Animals↗
Biomedical subjects
C Zwieb
Publications and source records attributed to C Zwieb.
DNA bending by negative regulatory proteins: Gal and Lac repressors.
The ability of two negative regulatory proteins, Gal and Lac repressors, to induce DNA bending was tested by the respective cloning of gal and lac operator DNA sequences into a sensitive vector, pBend2. pBend2 can generate a large number of DNA fragments in each of which a cloned operator is present in circularly permuted positions along the length of the DNA. Gel electrophoresis of these DNA fragments individually complexed to a repressor shows that the Gal repressor bends both of the gal operators, OE and OI. Similarly, the Lac repressor induces a bend to a lac operator DNA. In each case, the center of the average bent segment is located at or close to the dyad symmetry axis of the operator sequence. In view of these findings, we discuss how these negative regulatory proteins may function by a more dynamic mechanism than was perceived previously.
Base Sequence↗
Low resolution three-dimensional models of the 7SL RNA of the signal recognition particle, based on an intramolecular cross-link introduced by mild irradiation with ultraviolet light.
A number of intramolecular RNA-RNA cross-links were introduced into the human 7SL RNA by mild irradiation of a reconstituted signal recognition particle with ultraviolet light. Synthesis of radioactively labeled RNA was initiated in vitro from the T7 promoter. Smaller cross-linked complexes were generated by digestion of the RNA with RNase H in the presence of complementary deoxyoligonucleotides. Cross-linked complexes were separated from noncross-linked fragments by two-dimensional polyacrylamide gel electrophoresis. A close proximity between the stemloop around position 200 and the nucleotides at positions 67 to 76 was revealed by the absence of a characteristic oligonucleotide from the fingerprint of one of the complexes, suggesting a close spatial neighborhood between these two regions of the RNA. This and previous results, which described two different conformers of the RNA, were used to deduce two preliminary three-dimensional structure models of the 7SL RNA. The models differ in the base pairing scheme of the conserved core of the 7SL RNA.
Base Sequence↗
Isolation and characterization of a cDNA clone encoding the 19 kDa protein of signal recognition particle (SRP): expression and binding to 7SL RNA.
Signal recognition particle (SRP) consists of a 7SL RNA molecule and 6 protein subunits. We have isolated and characterized cDNA clones from human liver which encode the 19kDa protein subunit (SRP19). This subunit binds to the RNA directly and mediates binding of a second polypeptide, the 54kDa subunit which is involved in signal sequence recognition. Amino acid sequences deduced from the human cDNA sequence were identical to amino acid sequences of tryptic peptides from canine pancreatic SRP19. In vitro transcription and translation of the human cDNA resulted in a protein product the same size as canine SRP19 which could be immunoprecipitated by an antiserum raised against canine SRP19. SRP19 synthesized in a cell-free system specifically bound to 7SL RNA. The sequence of SRP19 is discussed with respect to its binding to 7SL RNA.
Amino Acid Sequence↗
Association of degradation and secretion of three chimeric polypeptides in Escherichia coli.
We investigated the stability of fusion proteins composed of the signal peptide of the heat-labile enterotoxin of Escherichia coli and three polypeptides: the bacterial cytoplasmic chloramphenicol acetyltransferase, the mouse dihydrofolate reductase, and human immune interferon. We demonstrate that these proteins are rapidly degraded as a result of being targeted to the secretion apparatus of E. coli, with the extent of degradation varying among the three fusion proteins. Four lines of experimental evidence are presented in support of this suggestion. First, the chimeric polypeptides containing a functional signal peptide were detected in low amounts in vivo. When a mutation was introduced in the signal peptide, resulting in lack of recognition by the secretion apparatus, the chimeric proteins accumulated at high levels in the cytoplasm of the cell. Second, both the wild-type and mutant polypeptides accumulated in a purified and reconstituted in vitro translation system from E. coli and were equally susceptible to digestion by an exogenous protease. Third, the chimeric polypeptides lacking the signal peptide accumulated in a stable form in vivo. Fourth, the precursors of the proteins containing a functional signal peptide accumulated in a secA ts mutant at the restrictive temperature when secretion was blocked, suggesting that degradation is tightly linked to the secretion apparatus.
Acetyltransferases↗
Protein translocation across wheat germ microsomal membranes requires an SRP-like component.
Different wheat germ extracts were tested for the presence of membranes capable of translocating and processing nascent secretory proteins. One lysate was found in which nascent prehuman-placental lactogen (phPL) was translocated and processed to mature human placental lactogen (hPL). Processing was found to occur concomitant with translocation across membranes. Translocation across the wheat germ membrane required a component which is similar to the mammalian signal recognition particle (SRP). It bound to DEAE-Sepharose, had a sedimentation coefficient of 11S and contained a 7S RNA. In addition to hPL, the plant protein zein and the bacterial protein beta-lactamase were translocated across and processed by wheat germ membranes. Transport was found to occur only co-translationally. Our results show that the wheat germ protein translocation system is similar to the mammalian one. Unlike the mammalian SRP, the particle purified from wheat germ did not arrest elongation of nascent secretory proteins.
Journal Article↗
Identification of dynamic sequences in the central domain of 7SL RNA.
The human 7SL RNA component of the signal recognition particle can be separated into four major conformers by nondenaturing polyacrylamide gel electrophoresis. We have investigated what sequences give 7SL RNA the property to exist in different conformations. The human 7SL gene 7L30.1 was mutagenized using the random linker insertion approach and twelve mutant genes carrying alterations in the central domain of 7SL RNA were characterized. Mutant RNAs were produced by in vitro transcription of the various templates and their electrophoretic behaviour was determined. Bases between positions 98 and 133 as well as 206 and 251 proved to be necessary for the 7SL RNA to be able to exist in alternative conformations, while changes at the positions 85 to 97, 144 to 166 and 252 to 266 did not abolish this property. The dynamic sequences are located in the "central T" in the secondary structure of the 7SL RNA. They are phylogenetically conserved and include bases which are homologeous to 5S ribosomal RNA. A dynamic core structure composed of the dynamic parts of the 7SL RNA is suggested. An attempt was made to define the different conformers present in the wild-type 7SL RNA. These alternative configurations could play a functional role during the initial stage of protein translocation across the membrane of the endoplasmic reticulum.
Base Sequence↗
Characterization of a collection of deletion mutants at the 3'-end of 16S ribosomal RNA of Escherichia coli.
Deletions were constructed in plasmid pKK3535 in the coding region for the 3'-end of E. coli 16S rRNA. The plasmid was cleaved with restriction endonuclease Hae2 under conditions favoring the production of single cut linear plasmid DNA and deletions were produced by digestion with exonuclease Bal31. Seven different deletions were isolated ranging in size from 90 to about 200 base pairs. Transcription of ribosomal DNA, processing of ribosomal RNA and incorporation of mutant rRNA into mutant particles was studied in UV-sensitive cells using a modified maxicell labeling procedure. The different mutants were missing defined features in the secondary structure of 16S rRNA and were characterized according to their stability, ability to be processed, sensitivity to colicin E3, and ability to bind ribosomal protein S1 and to interact with 50S subunits. These analyses show that the small stem and loop structure at positions 1350 to 1372 is necessary for the stability of rRNA. The deletion of the long terminal stem structure (1409-1491) in all mutant rRNAs does not block processing of the mutant rRNAs or S1 binding, although processing of the mutant rRNAs or S1 binding, although it does prevent the association of particles containing the mutant rRNA with 50S subunits.
Base Sequence↗
Point mutations in the 3' minor domain of 16S rRNA of E.coli.
Point mutations were produced near the 3' end of E. coli 16S rRNA by bisulfite mutagenesis in a 121 base loop-out (1385 to 1505) in a heteroduplex of wild type (pKK3535) and deletion mutant plasmids. Two highly conserved, single stranded regions flank an irregular helix (1409-1491) in the area studied. Only a single mutation was isolated in the flanking regions, a transition at C1402, (normally methylated on the base and ribose in rRNA). Mutations occurred throughout the irregular helix. All mutant rRNAs were processed and assembled into 30S subunits capable of interacting with 50S subunits. Growth rates ranged from faster to significantly slower than cells with the wild type transcript. In particular, mutations at C1467 or C1469 cause slow growth. These two transitions (in a bulge region within the helix) reduced the bulge by additional base pairing.
Base Sequence↗
The secondary structure of the 7SL RNA in the signal recognition particle: functional implications.
The secondary structure of the 7SL RNA in the signal recognition particle was determined by applying both a theoretical and an experimental approach. The compensatory base change approach was taken comparing the published sequences of human, Drosophila and Xenopus 7SL RNA's. The deduced secondary structure was confirmed by post-labeling of an RNase V1-nicked dog SRP with P32-pCp and RNA-ligase and analysis of the labeled RNA-fragments by non-denaturing/denaturing 2D polyacrylamide gel electrophoresis. Two interesting features in the secondary structure were revealed: Firstly, bases at positions 122 to 127 of the human 7SL RNA are not only able to pair with bases at positions 167 to 170, but also with a single-stranded region of the bases at positions 223 to 228, suggesting an alternative base pairing scheme for the 7SL RNA in all three organisms. In agreement with this finding, four different conformations were identified after transcription of the 7SL RNA from the genomic human clone. The involvement of the particular basepairing interaction postulated was confirmed by the analysis of a 7SL RNA deletion mutant (Sma1-409). Secondly, a significant sequence homology of the paired bases at positions 236 to 255 and 104 to 109 in 7SL RNA with bases in 5S ribosomal RNA at the positions 84 to 110 was noticed, suggesting that 5S ribosomal and 7SL RNA interact with the same target during protein biosynthesis. These findings are summarized by proposing a mechanism for the translational arrest of protein synthesis by the signal recognition particle using specific sequences and an alternative configuration in the 7SL RNA.
Animals↗
Oligonucleotide directed mutagenesis of Escherichia coli 5S ribosomal RNA: construction of mutant and structural analysis.
The ribosomal 5S RNA gene from the rrnB operon of E. coli was mutagenised in vitro using a synthetic oligonucleotide hybridised to M13 ssDNA containing that gene. The oligonucleotide corresponded to the 5S RNA sequence positions 34 to 51 and changed the guanosine at position 41 to a cytidine. The DNA containing the desired mutation was identified by dot blot hybridisation and introduced back into the plasmid pKK 3535 which contains the total rrnB operon in pBR 322. Plasmid coded 5S rRNA was selectively labeled with 32p using a modified maxi-cell system, and the replacement of guanosine G41 by cytidine was confirmed by RNA sequencing. The growth of cells containing mutant 5S rRNA was not altered by the base change, and the 5S rRNA was processed and incorporated into 50S ribosomal subunits and 70S ribosomes. The structure of wildtype and mutant 5S rRNA was compared by chemical modification of accessible guanosines with kethoxal and limited enzymatic digestion using RNase T1 and nuclease S1. These results showed that the wildtype and mutant 5S rRNA do not differ significantly in their structure. Furthermore, the formation, interconversion and stability of the two 5S rRNA A- and B-conformers are unchanged.
Base Sequence↗
Effects of site-directed mutations in the central domain of 16 S ribosomal RNA upon ribosomal protein binding, RNA processing and 30 S subunit assembly.
Using a multicopy plasmid encoding the Escherichia coli rrnB ribosomal RNA operon and the techniques of in vitro site-directed mutagenesis, we have introduced several small alterations into the central domain of 16 S rRNA, which encompasses nucleotides 560 to 890. Four of the rRNAs studied contained deletions and one contained an insertion. The altered small ribosomal subunit rRNAs were used to investigate relationships among 16 S rRNA processing, protein-16 S rRNA interactions and assembly of the 30 S ribosomal subunit. Analysis of plasmid-coded transcripts from maxicells revealed that products from wild-type 16 S rRNA genes were fully processed and assembled into mature 30 S subunits. Under the same conditions, the processing and assembly of transcripts derived from the mutant plasmids were severely impaired. In some instances, the mutations completely blocked both processes, while in other cases rRNA maturation and ribosome assembly were retarded, but not eliminated completely. In all cases, the mutations led to the accumulation of the 17 S precursor to 16 S rRNA. The mutant 17 S rRNAs were purified and incubated with various combinations of E. coli ribosomal proteins S6, S8, S15 and S18, which are known to bind to the central domain of 16 S rRNA. Ribonuclease digestion of the resulting protein-17 S rRNA complexes and fractionation of the products permitted detection of three distinct protein-RNA fragment complexes which contained S8, S8 + S15, or S6 + S8 + S15 + S18. Whereas wild-type 17 S rRNA was able to form all three of these complexes, deletion of nucleotides 693 to 721 or 822 to 874 abolished the interaction of S6 and S18, and removal of nucleotides 659 to 718 prevented the binding of S6, S15 and S18. In contrast, elimination of residue 614, or the presence of a 16-base insertion between nucleotides 614 and 615, had no significant effect on the binding of any of the four proteins tested. Together, our results demonstrate that 16 S rRNA maturation and 30 S subunit assembly are tightly coupled, and show that, in at least some cases, defects in these processes can be correlated with the inability of particular ribosomal proteins to associate with altered rRNA molecules. Moreover, we have confirmed the essentiality of certain rRNA sequences for the formation and/or stabilization of these protein-rRNA interactions.(ABSTRACT TRUNCATED AT 400 WORDS)
Centrifugation, Density Gradient↗
Point mutations in the middle of 16S ribosomal RNA of E. coli produced by deletion loop mutagenesis.
Using the plasmid pKK3535 , which contains the rrnB operon of Escherichia coli in pBR322, a deletion mutation was constructed which lacks bases 822 to 874 in the middle of the 16S ribosomal RNA. This results in an "amputation" of a very distinct stem and loop structure in the RNA. By forming a heteroduplex between the deletion plasmid and the original pKK3535 and by modifying the single-stranded deletion loops with bisulfite, we produced plasmids containing one or two base changes at positions 839, 840, 841, 867 or 876. The clustering of the mutations near the top of the stem, and the inability to get base changes at other positions, suggests that single alterations at particular positions severely affect the formation of a functional ribosome. The ability to recover mutations at these positions is not determined by the secondary structure of the DNA during bisulfite mutagenesis. Restriction enzyme analysis of 12 revertants from a slow growing mutant (altered at positions 839 and 876) shows that they did not compensate for the mutation by re-establishing the original wild type sequence.
Base Sequence↗
The structure of ribosomal RNA and its organization relative to ribosomal protein.
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Animals↗
Precise localisation of three intra-RNA cross-links in 23S RNA and one in 5S RNA, induced by treatment of Escherichia coli 50S ribosomal subunits with bis-(2-chloroethyl)-methylamine.
Treatment of E. coli 50S ribosomal subunits with low doses of bis-(2-chloroethyl)-methylamine ("nitrogen mustard") leads to formation of a number of intra-RNA and RNA-protein cross-links. After partial digestion of the cross-linked subunits with cobra venom nuclease, followed by destruction of the protein moiety with proteinase K, complexes containing the intra-RNA cross-links were isolated by two-dimensional gel electrophoresis. The individual complexes were subjected to oligonucleotide analysis, either directly or after a second partial digestion procedure using ribonuclease T1, and the cross-link sites determined. In 23S RNA, the cross-links found were between bases 763 and 1567, 1210 and 1236, 1482 and 1501; in 5S RNA, base 69 was cross-linked to base 107. The significance of these cross-links in relation to the three-dimensional organization of the ribosomal RNA is discussed.
Base Sequence↗
Secondary structure comparisons between small subunit ribosomal RNA molecules from six different species.
Secondary structure models are presented for three pairs of small subunit ribosomal RNA molecules. These are the 16S rRNA from E. coli cytoplasmic and Z. mays chloroplast ribosomes, the 18S rRNA from S. cerevisiae and X. laevis cytoplasmic ribosomes, and the 12S rRNA from human and mouse mitochondrial ribosomes. Using the experimentally-established secondary structure of the E. coli 16S rRNA as a basis, the models were derived both by searching for primary structural homology between the three classes of sequence (12S, 16S, 18S), and also by searching for compensating base changes in putative helical regions of each pair of sequences. The models support the concept that secondary structure of ribosomal RNA has been extensively conserved throughout evolution, differences in length between the three classes of sequence being accommodated in distinct regions of the molecules.
Animals↗
Secondary structure of the large subunit ribosomal RNA from Escherichia coli, Zea mays chloroplast, and human and mouse mitochondrial ribosomes.
Short base-paired RNA fragments, and fragments containing intra-RNA cross-links, were isolated from E. coli 23S rRNA or 50S ribosomal subunits by two-dimensional gel electrophoresis. The interactions thus found were used as a first basis for constructing a secondary structure model of the 23S rRNA. Sequence comparison with the 23S rDNA from Z. mays chloroplasts, as well as with the 16S (large subunit) rDNA from human and mouse mitochondria, enabled the experimental model to be improved and extrapolated to give complete secondary structures of all four species. The structures are organized in well-defined domains, with over 450 compensating base changes between the two 23S species. Some ribosomal structural "'switches" were found, one involving 5S rRNA.
Animals↗
Precise localization of the site of cross-linking between protein L4 and 23S ribonucleic acid induced by mild ultraviolet irradiation of Escherichia coli 50S ribosomal subunits.
Mild ultraviolet irradiation of Escherichia coli 50S ribosomal subunits causes a cross-linking reaction between protein and RNA, whose primary target is protein L4 [Möller, K., & Brimacombe, R. (1975) Mol. Gen. Genet. 141, 343]. Here we have determined the site of this cross-link both on L4 and on 23S RNA. For the site on the protein, a cross-linked protein-oligonucleotide complex was isolated and subjected to successive digestions with various proteases. In each case the peptide-oligonucleotide complexes formed were analyzed. It could clearly be shown that the cross-link site was contained within a characteristic peptide 16--20 amino acids long and that the amino acid concerned was the tyrosine residue at position 35 in the recently completed L4 sequence (M. Kimura and B. Wittmann-Liebold, personal communication). For the site on the RNA, a cross-linked L4--23S RNA complex was subjected to mild nuclease digestion, producing a range of L4--RNA fragments which were isolated with the help of a new two-dimensional gel electrophoresis system. Oligonucleotide analyses of these fragments, combined with successive nuclease digestions of the residual oligonucleotide attached to protein L4, established that the site of cross-linking was homogeneous, involving a uridine residue at position 615 in the recently determined 23S RNA sequence [Brosius, J., Dull, T. J., & Noller, H. F. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 201].
Amino Acid Sequence↗