Data Mining to Determine the Influence of Fluid Properties on the Integrity Test Values.

Eudralex volume 4, Annex 1, the European Union Good Manufacturing Practice for sterile products, requires that "The integrity of the sterilised filter should be verified before use" (1). Implicit in this requirement for a PUPSIT is the rationale that the sterilizing filter could sustain damage during sterilization or use (i.e.

Translation initiation complex formation with 30 S ribosomal particles mutated at conserved positions in the 3'-minor domain of 16 S RNA.

Escherichia coli 30 S ribosomal subunits containing in vitro (phage T7 RNA polymerase-generated) 16 S rRNA, both wild-type and mutant, were examined by toeprinting. These synthetic particles were used to compare the effects of the absence of base modification and of specific nucleotide substitutions in conserved sequence regions of the RNA on the assembly of mRNA, tRNAs and 30 S particles into a translational initiation complex. Initiation factor-3-dependent selection of tRNA(fMet) from a mixture of tRNA(fMet) and tRNA(Phe) occurred with all particles, although 20 times less initiation factor-3 was needed for the synthetic particles, including the mutants.

Functional effects of base changes which further define the decoding center of Escherichia coli 16S ribosomal RNA: mutation of C1404, G1405, C1496, G1497, and U1498.

The existence and functional importance of the tertiary base pair G1401:C1501, which brings together two universally present and highly sequence-conserved single-stranded segments of small subunit ribosomal RNA, was proven recently by mutational analysis [Cunningham, P. R., Nurse, K.

Chemical evidence for domain assembly of the Escherichia coli 30S ribosome.

A fragment of 16S RNA corresponding to most of the 5'-domain (residues 1-526) was prepared by in vitro run-off transcription. When this fragment was incubated with a mixture of 30S proteins under conditions known to result in the in vitro assembly of a complete, functional 30S ribosome from a full-length transcript, a discrete 16S particle was formed. This particle contained near stoichiometric amounts of ribosomal proteins S4, S16, S17, and S20.

Interaction between the two conserved single-stranded regions at the decoding site of small subunit ribosomal RNA is essential for ribosome function.

Formation of the tertiary base pair G1401:C1501, which brings together two universally present and highly sequence-conserved single-stranded segments of small subunit ribosomal RNA, is essential for ribosome function. It was previously reported that mutation of G1401 inactivated all in vitro functions of the ribosome [Cunningham et al. (1992) Biochemistry 31, 7629-7637].

G1401: a keystone nucleotide at the decoding site of Escherichia coli 30S ribosomes.

16S ribosomal RNA contains three highly conserved single-stranded regions. Centrally located in one of these regions is the C1400 residue. Zero-length cross-linking of this residue to the anticodon of ribosome-bound tRNA showed that it was at or near the ribosomal decoding site [Ehresmann, C.

A paradigm for local conformational control of function in the ribosome: binding of ribosomal protein S19 to Escherichia coli 16S rRNA in the presence of S7 is required for methylation of m2G966 and blocks methylation of m5C967 by their respective methyltransferases.

We have partially purified two 16S rRNA-specific methyltransferases, one of which forms m2G966 (m2G MT), while the other one makes m5C967 (m5C MT). The m2G MT uses unmethylated 30S subunits as a substrate, but not free unmethylated 16S rRNA, while the m5C MT functions reciprocally, using free rRNA but not 30S subunits (Nègre, D., Weitzmann, C.

SP6 RNA polymerase stutters when initiating from an AAA... sequence.

The 16S ribosomal RNA gene of Escherichia coli was placed under the transcriptional control of consensus and modified T7 promoters and a modified SP6 promoter. Both T7 and SP6 polymerases faithfully transcribed the coding sequence (beginning at the +1 position) of each construct, although SP6 polymerase was five-fold more effective than T7 polymerase in initiating with the AAAUUG..

The absence of modified nucleotides affects both in vitro assembly and in vitro function of the 30S ribosomal subunit of Escherichia coli.

16S RNA of Escherichia coli lacking all post-transcriptional modifications and with 5'-termini of pppGGGAGA-, pppGAA-, pppAAA-, and pAAA- were prepared by in vitro transcription of appropriately engineered plasmids with T7 or SP6 RNA polymerases. These synthetic versions of 16S RNA were compared with natural 16S RNA for their ability to reconstitute 30S ribosomal subunits in vitro using varied conditions for both the isolation of the RNA and for reconstitution. Under all conditions studied, natural 16S RNA assembled correctly, as judged by velocity centrifugation comparison with an internal standard of native 30S particles, and the recovered ribosomes were 80-100% as active as native 30S ribosomes in initiation complex formation, P site binding of AcVal-tRNA, A site binding of Phe-tRNA, and formation of the first peptide bond.

Site-specific mutation of the conserved m6(2)A m6(2)A residues of E. coli 16S ribosomal RNA. Effects on ribosome function and activity of the ksgA methyltransferase.

In vitro synthesis of mutant 16S RNA and reconstitution with ribosomal proteins into a mutant 30S ribosome was used to make all possible single base changes at the universally conserved A1518 and A1519 residues. All of the mutant RNAs could be assembled into a ribosomal subunit which sedimented at 30 S and did not lack any of the ribosomal proteins. A series of in vitro tests of protein synthesis ability showed that all of the mutants had some activity.

Cloning, in vitro transcription, and biological activity of Escherichia coli 23S ribosomal RNA.

The 23S rRNA gene was excised from the rrnB operon of pKK3535 and ligated into pUC19 behind the strong class III T7 promoter so that the correct 5' end of mature 23S RNA was produced upon transcription by T7 RNA polymerase. At the 3' end, generation of a restriction site for linearization required the addition of 2 adenosine residues to the mature 23S sequence. In vitro runoff transcripts were indistinguishable from natural 23S RNA in size on denaturing gels and in 5'-terminal sequence.

Reconstitution of Escherichia coli 50S ribosomal subunits containing puromycin-modified L23: functional consequences.

In previous work we have shown that both puromycin [Weitzmann, C. J., & Cooperman, B.

Protein estimation by the product of integrated peak area and flow rate.

A convenient method for protein estimation is described, making use of uv detectors and peak integrators that are standard equipment on modern high-performance liquid chromatographs to determine the product of integrated peak area and flow rate of eluting protein at 214 nm (AF214). We demonstrate that AF214 is proportional to the amount of eluted protein and describe two approaches for calibrating the integrator, by quantitative amino acid analysis and by determining the elution yield of a known amount of applied protein, allowing direct estimation of protein from AF214. Both approaches yield similar results.

In vitro methylation of Escherichia coli 16S ribosomal RNA and 30S ribosomes.

Treatment of synthetic 30S particles lacking all of the normally methylated nucleotides with S-adenosyl-[3H]methionine and either an S100 or ribosomal high salt wash extract resulted in ribosome-dependent incorporation of [3H]methyl groups into trichloroacetic acid-insoluble material. No incorporation was observed when naturally methylated isolated 30S particles were used, showing that methylation at unnatural sites did not occur. Enzymatic hydrolysis of the labeled RNA to nucleosides followed by HPLC analysis identified the [3H]methylated residues.

Effect of point mutations in the decoding site (C1400) region of 16S ribosomal RNA on the ability of ribosomes to carry out individual steps of protein synthesis.

In order to probe the relationship between structure and function of the ribosome, an in vitro system [Denman et al. (1988) Biochemistry (preceding paper in this issue)] was used to make a series of base changes around C1400, a residue known to be at the decoding site. Replacement of C1400 by U, A, or G, deletion of single bases at and to either side of C1400, and insertion of C or U next to C1400 were done.

In vitro assembly of 30S and 70S bacterial ribosomes from 16S RNA containing single base substitutions, insertions, and deletions around the decoding site (C1400).

An in vitro system developed for the site-specific mutagenesis of 16S RNA of Escherichia coli ribosomes [Krzyzosiak et al. (1987) Biochemistry 26, 2353-2364] was used to make 10 single base changes around C1400, a residue known to be at the decoding site. C1400 was replaced by U, A, or G, five single base deletions at and to either side of C1400 were made, and C or U was inserted next to C1400.

The role of 16S RNA in ribosome function: single base alterations and their effect on in vitro protein synthesis.

An in vitro system developed for the site-specific mutagenesis of 16S RNA of Escherichia coli ribosomes (Krzyzosiak et al., Biochemistry 26, 2353-2364, 1987) was used to make 10 single base changes around C1400, the residue known to be at the decoding site. C1400 was replaced by U, A, or G, 5 single base deletions at and to either side of C1400 were made, and C or U was inserted next to C1400.

On the structural specificity of puromycin binding to Escherichia coli ribosomes.

We have examined the structural specificity of the puromycin binding sites on the Escherichia coli ribosome that we have previously identified [Nicholson, A. W., Hall, C.

Application of high-performance liquid chromatography to the reconstitution of ribosomal subunits.

We are currently utilizing reversed-phase high-performance liquid chromatography (RP-HPLC) in reconstitution experiments designed to study the structure and function of Escherichia coli ribosomes. The applications of RP-HPLC in these experiments include: (a) preparation of individual proteins or groups of proteins on a milligram scale for reconstitution pools, (b) analysis of the protein stoichiometry of reconstituted subunits, (c) determination of the extent and specificity of modification of proteins extracted from ribosomal subunits which have been subjected to chemical modification, and (d) resolution of modified forms of proteins S14 and L23 from the corresponding unmodified proteins. Proteins prepared by RP-HPLC from 30S and 50S ribosomal subunits were found to reconstitute into 30S and 50S subunits respectively, as well as into slower sedimenting particles.

Reversed-phase high-performance liquid chromatography of Escherichia coli ribosomal proteins. Characteristics of the separation of a complex protein mixture.

We have previously reported the application of reversed-phase high-performance liquid chromatography (RP-HPLC) to the separation of Escherichia coli ribosomal proteins (A. R. Kerlavage, L.