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R A Zimmermann

Publications and source records attributed to R A Zimmermann.

At least 37 records · Page 2Linked to original sources
PubMed1992

Photochemical cross-linking of the anticodon loop of yeast tRNA(Phe) to 30S-subunit protein S7 at the ribosomal A and P sites.

Yeast tRNA(Phe), containing the photoreactive nucleoside 2-azidoadenosine at position 37 within the anticodon loop, has been cross-linked to the aminoacyl-tRNA (A) and peptidyl-tRNA (P) binding sites of the Escherichia coli ribosome. The 30S subunit was exclusively labeled in each case, and cross-linking occurred to both protein and 16S rRNA. Electrophoretic and immunological analyses demonstrated that S7 was the only 30S-subunit protein covalently attached to the tRNA. However, digestion of the A and P site-labeled S7 with trypsin revealed a unique pattern of cross-linked peptide(s) at each site. Thus, while the anticodon loop of tRNA is in close proximity to protein S7 at both the A and P sites, it neighbors a different portion of the protein molecule in each. The placement of the aminoacyl- and peptidyl-tRNA binding sites is discussed in relationship to recent models of the 30S ribosomal subunit.

Anticodon↗
PubMed1992

Mutagenesis of ribosomal protein S8 from Escherichia coli: defects in regulation of the spc operon.

The structural features of Escherichia coli ribosomal protein S8 that are involved in translational regulation of spc operon expression and, therefore, in its interaction with RNA have been investigated by use of a genetic approach. The rpsH gene, which encodes protein S8, was first inserted into an expression vector under the control of the lac promoter and subsequently mutagenized with methoxylamine or nitrous acid. A screening procedure based on the regulatory role of S8 was used to identify mutants that were potentially defective in their ability to associate with spc operon mRNA and, by inference, 16S mRNA. In this way, we isolated 39 variants of the S8 gene containing alterations at 34 different sites, including 37 that led to single amino acid substitutions and 2 that generated premature termination codons. As the mutations were distributed throughout the polypeptide chain, our results indicate that amino acid residues important for the structural integrity of the RNA-binding domain are not localized to a single segment. Nonetheless, the majority were located within three short sequences at the N terminus, middle, and C terminus that are phylogenetically conserved among all known eubacterial and chloroplast versions of this protein. We conclude that these sites encompass the main structural determinants required for the interaction of protein S8 with RNA.

Bacterial Proteins↗
PubMed1990

Probing tRNA binding sites on the Escherichia coli 30 S ribosomal subunit with photoreactive analogs of the anticodon arm.

Two analogs of the anticodon arm of yeast tRNAPhe (residues 28-43), in which G43 was replaced by the photoreactive nucleosides 2-azidoadenosine and 8-azidoadenosine, have been used to create 'zero-length' cross-links to ribosomal components at the peptidyl-tRNA binding site (P site) of 30 S subunits from the Escherichia coli ribosome. To prepare the analogs, 2-azidoadenosine and 8-azidoadenosine bisphosphates were first ligated to the 3' end of the anticodon-containing dodecanucleotide ACmUGmAAYA psi m5CUG from yeast tRNAPhe. The trinucleotide CAG was then joined to the 5' end of the resulting tridecanucleotide in a subsequent ligation. Both analogs bound to poly(U)-programmed 30 S subunits with affinities similar to that of the unmodified anticodon arm from yeast tRNAPhe. Irradiation of noncovalent complexes containing the photolabile analogs, poly(U) and 30 S ribosomal subunits with 300 nm light led to the covalent attachment of the anticodon arms to proteins S13 and S19. Further analysis revealed that S13 accounted for about 80%, and S19 for about 20%, of the cross-linked material. Labeling of these two proteins with 'zero-length' cross-linking probes provides useful information about the location and orientation of P site-bound tRNA on the ribosome and permits a test of recently proposed models of the three-dimensional structure of the 30 S subunit.

Adenosine↗
PubMed1990

Organization and expression of the 16S, 23S and 5S ribosomal RNA genes from the archaebacterium Thermoplasma acidophilum.

To elucidate the organization of the transcription units encoding the 16S, 23S and 5S rRNAs in the archaebacterium Thermoplasma acidophilum, the nucleotide sequences flanking the three rRNA genes were determined, and the 5' and 3' termini of the rRNA transcripts were mapped by primer extension and nuclease S1 protection. The results show that each of the rRNAs is transcribed separately, consistent with the lack of physical proximity among them in the T. acidophilum genome. The transcription initiation sites are preceded at an interval of approximately 25 base pairs by conserved A + T-rich sequences of the form CTTATATA, which strongly resemble the archaebacterial promoter consensus, TTTAT/AATA. In all three cases, transcription termination occurs within T-rich tracts just downstream from inverted repeats which can be folded into relatively stable stem-loop structures. While no partially processed intermediates of the 16S or 5S rRNA transcripts were detected, the 23S rRNA transcript appears to be processed by a RNase III-like activity prior to final maturation. This is the only organism known in the prokaryotic world in which the 16S, 23S and 5S rRNAs are all expressed from separate transcription units.

Base Sequence↗
PubMed1989

Preparation of 2-azidoadenosine 3',5'-[5'-32P]bisphosphate for incorporation into transfer RNA. Photoaffinity labeling of Escherichia coli ribosomes.

2-Azidoadenosine was synthesized from 2-chloroadenosine by sequential reaction with hydrazine and nitrous acid and then bisphosphorylated with pyrophosphoryl chloride to form 2-azidoadenosine 3',5'-bisphosphate. The bisphosphate was labeled in the 5'-position using the exchange reaction catalyzed by T4 polynucleotide kinase in the presence of [gamma-32P]ATP. Polynucleotide kinase from a T4 mutant which lacks 3'-phosphatase activity (ATP:5'-dephosphopolynucleotide 5'-phosphotransferase, EC 2.7.1.78) was required to facilitate this reaction. 2-Azidoadenosine 3',5'-[5'-32P]bisphosphate can serve as an efficient donor in the T4 RNA ligase reaction and can replace the 3'-terminal adenosine of yeast tRNAPhe with little effect on the amino acid acceptor activity of the tRNA. In addition, we show that the modified tRNAPhe derivative can be photochemically cross-linked to the Escherichia coli ribosome.

Adenosine Diphosphate↗
PubMed1989

Photochemical labeling of bovine pancreatic ribonuclease A with 8-azidoadenosine 3',5'-bisphosphate.

A simple method has been developed for the preparation of 5'-32P-labeled 8-azidoadenosine 3',5'-bisphosphate (p8N3Ap) for use in photoaffinity labeling studies. Irradiation of a complex between p8N3Ap and bovine pancreatic ribonuclease A (RNase A) with light of 300-350 nm led to the covalent attachment of the nucleotide to the enzyme. RNase A could also be labeled in the dark with prephotolyzed p8N3Ap. In either case, the nucleotide reacted with the same tryptic peptide, encompassing amino acids 67-85 of the protein. The site of labeling was determined to be either Thr-78 or Thr-82, both of which are close to or at the pyrimidine binding site of the enzyme. This result is consistent with recent nuclear magnetic resonance and X-ray studies which indicate that 8-substituted adenine nucleotides interact with the pyrimidine binding site of RNase A.

Adenosine Diphosphate↗
PubMed1989

Labeling the peptidyltransferase center of the Escherichia coli ribosome with photoreactive tRNA(Phe) derivatives containing azidoadenosine at the 3' end of the acceptor arm: a model of the tRNA-ribosome complex.

Photoreactive derivatives of yeast tRNA(Phe) containing 2-azidoadenosine (2N3A) at position 73 or 76 have been crosslinked to the peptidyl site of Escherichia coli ribosomes. Covalent tRNA-ribosome attachment was dependent upon the replacement of adenosine by 2N3A in the tRNA, irradiation with 300-nm light, and the presence of poly(U). In all cases, the modified tRNAs became crosslinked exclusively to 50S ribosomal subunits. While the tRNA derivative containing 2N3A at position 73 labeled only protein L27, that containing 2N3A at position 76 labeled proteins L15, L16, and L27 as well as a segment of the 23S rRNA. The site of crosslinking in the rRNA was identified as guanosine-1945, which lies within a highly conserved sequence adjacent to a number of modified bases and has not until now been identified at the peptidyltransferase center. On the basis of these results, and previously reported crosslinks from tRNA containing 8-azidoadenosine in the 3'-terminal -A-C-C-A sequence [Wower, J., Hixson, S. S. & Zimmermann, R. A. (1988) Biochemistry 27, 8114-8121], we propose a model for the arrangement of tRNA molecules at the peptidyl and aminoacyl sites that is consistent with most of the information available about the location of the peptidyltransferase center and the decoding domain of the E. coli ribosome.

Acyltransferases↗
PubMed1989

The structure and organization of the 16S ribosomal RNA gene from the archaebacterium Thermoplasma acidophilum.

The complete nucleotide sequence of the 16S rRNA gene from Thermoplasma acidophilum, as well as its 5' and 3' flanking regions, were determined by the dideoxynucleotide chain termination method. The 16S rRNA gene encodes 1471 nucleotides. The primary and secondary structures of T. acidophilum 16S rRNA both exhibit typical archaebacterial features. The sequence appears to be more closely related to 16S rRNAs of the methanogen--halophile group than to those of the thermoacidophile group. Secondary-structure comparisons generally support this relationship, although there are several examples in which the single-stranded loops in particular helices of T. acidophilum 16S rRNA more strongly resemble their counterparts in the 16S rRNA of Sulfolobus solfataricus, a member of the thermoacidophile group. In contrast to the polycistronic rRNA operons found in most organisms, the three rRNA genes from T. acidophilum occur in only a single copy per genome and appear to be physically unlinked. Consistent with this, the 16S rRNA gene is flanked by putative promoter and terminator sequences that are comparable to the transcription control signals from other archaebacterial genes. The sequence TATATATA, which is very similar to the archaebacterial promoter consensus TTTAT/AATA, is located 18 bases before the probable site of transcription initiation, TGCACAT. There is a potential transcription termination site immediately downstream from the gene that consists of a relatively stable stem and loop structure followed by stretches of Tresidues.

Amino Acid Sequence↗
PubMed1988

Interaction of Escherichia coli ribosomal protein S8 with its binding sites in ribosomal RNA and messenger RNA.

The ability of ribosomal protein S8 from Escherichia coli to interact with 12 variants of its 16 S rRNA binding site, as well as with a regulatory sequence within spc operon mRNA, has been assessed. Single-site alterations were introduced into the appropriate segment of the E. coli 16 S rRNA gene by mutagenesis in vitro. Their effects on S8-rRNA interaction were measured via a filter-binding assay, utilizing S8 binding sites transcribed in vitro from the altered 16 S rRNA gene fragments. Of the 12 rRNA mutants, six were unable to bind S8. Significantly, five of these occur within a small, phylogenetically conserved internal loop, defined by nucleotides 596-597 and 641-643, suggesting that this structure plays a major role in S8-16 S rRNA recognition. The reduced affinity of S8 for its binding site in these cases was closely correlated with growth defects that resulted from expression of the same mutations in vivo. Alterations at other positions in the S8 binding site had little influence on complex formation or cell growth, as long as they did not disrupt rRNA secondary structure. The specific interaction of S8 with a segment of the spc operon mRNA containing a putative site of translational feedback regulation was demonstrated using appropriate in vitro transcripts in conjunction with the filter-binding assay. The apparent association constant for the S8-mRNA interaction was determined to be approximately 5 x 10(6) M-1, about five times lower than for the interaction of S8 with wild-type 16 S rRNA. The structure of the regulatory binding site, determined by sequence analysis of spc operon mRNA protected by S8 from RNase digestion, was found to contain all of the characteristic features of the 16 S rRNA binding site, demonstrating that the protein associates with structurally similar domains in both RNAs.

Base Sequence↗
PubMed1988

Photochemical cross-linking of yeast tRNA(Phe) containing 8-azidoadenosine at positions 73 and 76 to the Escherichia coli ribosome.

The 3'-terminal -A-C-C-A sequence of yeast tRNA(Phe) has been modified by replacing either adenosine-73 or adenosine-76 with the photoreactive analogue 8-azidoadenosine (8N3A). The incorporation of 8N3A into tRNA(Phe) was accomplished by ligation of 8-azidoadenosine 3',5'-bisphosphate to the 3' end of tRNA molecules which were shortened by either one or four nucleotides. Replacement of the 3'-terminal A76 with 8N3A completely blocked aminoacylation of the tRNA. In contrast, the replacement of A73 with 8N3A has virtually no effect on the aminoacylation of tRNA(Phe). Neither substitution hindered binding of the modified tRNAs to Escherichia coli ribosomes in the presence of poly(U). Photoreactive tRNA derivatives bound noncovalently to the ribosomal P site were cross-linked to the 50S subunit upon irradiation at 300 nm. Nonaminoacylated tRNA(Phe) containing 8N3A at either position 73 or position 76 cross-linked exclusively to protein L27. When N-acetylphenylalanyl-tRNA(Phe) containing 8N3A at position 73 was bound to the P site and irradiated, 23S rRNA was the main ribosomal component labeled, while smaller amounts of the tRNA were cross-linked to proteins L27 and L2. Differences in the labeling pattern of nonaminoacylated and aminoacylated tRNA(Phe) containing 8N3A in position 73 suggest that the aminoacyl moiety may play an important role in the proper positioning of the 3' end of tRNA in the ribosomal P site. More generally, the results demonstrate the utility of 8N3A-substituted tRNA probes for the specific labeling of ribosomal components at the peptidyltransferase center.

Adenosine↗
PubMed1988

Mutations within the decoding site of Escherichia coli 16S rRNA: growth rate impairment, lethality and intragenic suppression.

Several C----U transitions and small deletions were introduced into the conserved region centered on base C1400 in Escherichia coli 16S rRNA by in vitro mutagenesis. The mutations were placed within rrnB operons on multicopy plasmids under the transcriptional regulation of either the normal rrnB P1P2 promoters or the temperature-inducible PL promoter from bacteriophage lambda and introduced into E. coli hosts. When expressed from the P1P2 promoters, several of the mutant 16S rRNAs impaired cell growth while others, including one in which U replaced C at position 1400 within the ribosomal decoding site, had little or no effect on cell doubling time. However, C----U transitions at positions 1395 and 1407, as well as the deletion of C1400, appeared to render their hosts inviable. Cells in which these mutations were expressed from the lambdaPL promoter died within four generations after induction. Unexpectedly, the lethal phenotype was suppressed intragenically by replacement of G1505 with A, C or U. Suppression may alleviate a functional defect in 30S subunits containing the U1395, U1407 or deltaC1400 mutations.

Base Sequence↗
PubMed1986

Site-directed mutagenesis of the binding site for ribosomal protein S8 within 16S ribosomal RNA from Escherichia coli.

Twelve specific alterations have been introduced into the binding site for ribosomal protein S8 in Escherichia coli 16S rRNA. Appropriate rDNA segments were first cloned into bacteriophage M13 vectors and subjected to bisulfite and oligonucleotide-directed mutagenesis in vitro. Subsequently, the mutagenized sequences were placed within the rrnB operon of plasmid pNO1301 and the mutant plasmids were used to transform E. coli recipients. The growth rates of cells containing the mutant plasmids were determined and compared with that of cells containing the wild-type plasmid. Only those mutations which occurred at highly conserved positions, or were expected to disrupt the secondary structure of the binding site, increased the doubling time appreciably. The most striking changes in growth rate resulted from mutations that altered a small internal loop within the S8 binding site. This structure is phylogenetically conserved in prokaryotic 16S rRNAs and may play a direct role in S8-16S rRNA recognition and interaction.

Base Sequence↗
PubMed1985

Photochemical cross-linking of tRNA1Arg to the 30S ribosomal subunit using aryl azide reagents attached to the anticodon loop.

The 2-thiocytidine residue at position 32 of tRNA1Arg from Escherichia coli was modified specifically with three photoaffinity reagents of different lengths, and the corresponding N-acetylarginyl-tRNA1Arg derivatives were cross-linked to the P site of E. coli 70S ribosomes by irradiation. Covalent attachment was dependent upon the presence of a polynucleotide template and exposure to light of the appropriate wavelength. From 4% to 6% of the noncovalently bound tRNA became cross-linked to the ribosome as a result of photolysis, and attachment to the P site was confirmed by the reactivity of arginine in the covalent complexes toward puromycin. Analysis of the irradiated ribosomes by sucrose-gradient sedimentation at low Mg2+ concentration revealed that the tRNA was associated exclusively with the 30S subunit in all cases. Two of the N-acetylarginyl-tRNA1Arg derivatives were attached primarily to ribosomal proteins whereas the third was cross-linked mainly to 16S RNA. Partial RNase digestion of the latter complex demonstrated that the tRNA had become attached to the 3' third of the rRNA molecule. In addition, the tRNA-rRNA bond was shown to be susceptible to cleavage by hydroxylamine and mercaptoethanol.

Anticodon↗
PubMed1985

Covalent cross-linking of tRNAGly1 to the ribosomal P site via the dihydrouridine loop.

The dihydrouracil residue at position 20 of Escherichia coli tRNAGly1 has been replaced by the photoaffinity reagent, N-(4-azido-2-nitrophenyl)glycyl hydrazide (AGH). The location of the substituent was confirmed by the susceptibility of the modified tRNA to cleavage with aniline. When N-acetylglycyl-tRNAGly1 derivatized with AGH was bound noncovalently to the P site of E. coli 70 S ribosomes, 5-6% on average was photochemically cross-linked to the ribosomal particles in a reaction requiring poly(G,U), irradiation and the presence of the AGH label in the tRNA. Approximately two-thirds of the covalently attached tRNA was associated with 16 S RNA in the 30 S subunit. This material was judged to be in the P site by the criterion of puromycin reactivity. As partial RNAase digestion of the tRNA-16 S RNA complex produced labeled fragments from both 5' and 3' segments of the rRNA, there appeared to be more than one site of cross-linking in the 30 S subunit. The small amount of N-acetylglycyl-tRNAGly1 associated with the 50 S subunit was also linked mainly to rRNA, but it was not puromycin-reactive.

Affinity Labels↗
PubMed1985

Isolation and cell-free synthesis of variant surface glycoproteins from Trypanosoma congolense.

Two Trypanosoma congolense stocks, 1/148 FLY and TREU 921, were cloned in A/J strain mice immunosuppressed with cyclophosphamide. The cloned populations, AmNat 1.1 and AmNat 3.1, each characterized by a different variant antigen type, were checked for homogeneity by the indirect fluorescent antibody test using 6-day antisera developed in rabbits. The variant surface glycoproteins (VSGs) from both AmNat clones were purified to homogeneity. Electrophoresis on sodium dodecyl sulfate (SDS)-polyacrylamide gradient gels revealed that the apparent Mr values of the two VSGs were 51 700 (AmNat 1.1) and 49 900 (AmNat 3.1). Monospecific antisera prepared in rabbits to each VSG were used to confirm the homogeneity of the clones by the indirect fluorescent antibody test. The VSGs were susceptible to endoglycosidase H digestion, indicating the presence of high-mannose type oligosaccharides in these glycoproteins. The apparent Mr values of the endoglycosidase H-digested VSGs were 48 800 and 46 900 for AmNat 1.1 and 3.1, respectively. Poly(A+)-enriched RNA isolated from each clone was assayed for template activity using a mRNA-dependent rabbit reticulocyte lysate for in vitro protein synthesis. Radioactively labeled polypeptides were initially characterized by SDS-polyacrylamide gradient gel electrophoresis and visualized by fluorography. VSG-specific translation products were immunoprecipitated with IgGs isolated from the homologous monospecific antisera and analyzed on SDS-polyacrylamide gradient gels. The apparent Mr values for the AmNat 1.1 and 3.1 precursor VSGs synthesized in vitro were 39 000 and 43 000, respectively.

Animals↗
PubMed1984

Interaction of ribosomal proteins S6, S8, S15 and S18 with the central domain of 16 S ribosomal RNA from Escherichia coli.

The co-operative interaction of 30 S ribosomal subunit proteins S6, S8, S15 and S18 with 16 S ribosomal RNA from Escherichia coli was studied by (1) determining how the binding of each protein is influenced by the others and (2) characterizing a series of protein-rRNA fragment complexes. Whereas S8 and S15 are known to associate independently with the 16 S rRNA, binding of S18 depended upon S8 and S15, and binding of S6 was found to require S8, S15 and S18. Ribonucleoprotein (RNP) fragments were derived from the S8-, S8/S15- and S6/S8/S15/S18-16 S rRNA complexes by partial RNase hydrolysis and isolated by electrophoresis through Mg2+-containing polyacrylamide gels or by centrifugation through sucrose gradients. Identification of the proteins associated with each RNP by gel electrophoresis in the presence of sodium dodecyl sulfate demonstrated the presence of S8, S8 + S15 and S6 + S8 + S15 + S18 in the corresponding fragment complexes. Analysis of the rRNA components of the RNP particles confirmed that S8 was bound to nucleotides 583 to 605 and 624 to 653, and that S8 and S15 were associated with nucleotides 583 to 605, 624 to 672 and 733 to 757. Proteins S6, S8, S15 and S18 were shown to protect nucleotides 563 to 605, 624 to 680, 702 to 770, 818 to 839 and 844 to 891, which span the entire central domain of the 16 S rRNA molecule (nucleotides 560 to 890). The binding site for each protein contains helical elements as well as single-stranded internal loops ranging in size from a single bulged nucleotide to 20 bases. Three terminal loops and one stem-loop structure within the central domain of the 16 S rRNA were not protected in the four-protein complex. Interestingly, bases within or very close to these unprotected regions have been shown to be accessible to chemical and enzymatic probes in 30 S subunits but not in 70 S ribosomes. Furthermore, nucleotides adjacent to one of the unprotected loops have been cross-linked to a region near the 3' end of 16 S rRNA. Our observations and those of others suggest that the bases in this domain that are not sequestered by interactions with S6, S8, S15 or S18 play a role involved in subunit association or in tertiary interactions between portions of the rRNA chain that are distant from one-another in the primary structure.(ABSTRACT TRUNCATED AT 400 WORDS)

Base Sequence↗
PubMed1984

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↗