Genetic approaches to studying protein synthesis: effects of mutations at Psi516 and A535 in Escherichia coli 16S rRNA.

A genetic system for the study of ribosomal RNA function and structure was developed. First, the ribosome binding sequence of the chloramphenicol acetyltransferase gene and the message binding sequence of 16S ribosomal RNA were randomly mutated and alternative highly functional sequences were selected and characterized. From this set of mutants, a single clone was chosen and subjected to a second round of mutagenesis to optimize the specificity of the system. In the resulting system, plasmid-encoded ribosomes efficiently and exclusively translate specific mRNA containing the appropriate ribosome binding sequences. This system allows facile isolation and analysis of mutations that would normally be lethal and allows direct selection of rRNA mutants with predetermined levels of ribosome function. The system was used to examine the effects of mutations at the sole pseudouridine (Psi) in Escherichia coli 16S rRNA which is located at position 516 of the conserved 530 loop. The nucleotide opposite Psi516 in the hairpin, A535, was also mutated. The data show that a pyrimidine (Psi or C) is required at position 516, while substitutions at position 535 reduce ribosome function by < 50%. A requirement for base pair formation between Psi516 and A535 was not indicated.

Structure and function of the conserved 690 hairpin in Escherichia coli 16 S ribosomal RNA. II. NMR solution structure.

The solution structure of the conserved 690 hairpin from Escherichia coli 16 S rRNA was determined by NMR spectroscopy. The 690 loop is located at the surface of the 30 S subunit in the platform region and has been implicated in interactions with P-site bound tRNA, E-site mRNA, S11 binding, IF3 binding, and in RNA-RNA interactions with the 790 loop of 16 S rRNA and domain IV of 23 S rRNA. The structure reveals a novel sheared type G690.U697 base-pair with a single hydrogen bond from the G690 amino to U697-04. G691 and A696 also form a sheared pair and U692 forms a U-turn with an H-bond to the A695 non-bridging phosphate oxygen. The sheared pairs and U-turn result in the continuous single-stranded stacking of five residues from 6693 to U697 with their Watson-Crick functional groups exposed in the minor groove. The overall fold of the 690 hairpin is similar to the anticodon loop of tRNA. The structure provides an explanation for chemical protection patterns in the loop upon interaction with tRNA, the 50 S subunit, and S11. In vivo genetic studies demonstrate the functional importance of the motifs observed in the solution structure of the 690 hairpin.

Structure and function of the conserved 690 hairpin in Escherichia coli 16 S ribosomal RNA. III. Functional analysis of the 690 loop.

An instant-evolution experiment was performed on the eight nucleotides comprising the loop region of the 690 hairpin in Escherichia coli 16 S ribosomal RNA. Positions 690 to 697 were randomly mutated and 101 unique functional mutants were isolated, sequenced and analyzed for function in vivo. Non-random nucleotide distributions were observed at each of the mutated positions except 693 and 694. Nucleotide identity significantly affected ribosome function at positions 690, 695, 696 and 697. Pyrimidines were absent at position 696 in the instant-evolution pool as were C at position 691 and G at position 697. A highly significant covariation was observed between nucleotides 690 and 697. No functional double mutants at positions 691 and 696 were obtained from the instant-evolution pool. In our NMR structure of the 690 loop, both the G690.U697 and G691.A696 form sheared hydrogen-bonded mismatches. To further examine the functional constraints between these paired nucleotides, one set of site-directed mutations was constructed at positions 690:697 and another set was constructed at positions 691:696. Functional analysis of the site-directed mutants is consistent with our instant-evolution findings and revealed constraints on the placement of specific functional groups observed in the NMR structure. Ten instant-evolution mutants were isolated that are more functional than the wild-type. Hyperactivity in these mutants correlates with a single mutation at position 693.

Aerobic activity of Escherichia coli alcohol dehydrogenase is determined by a single amino acid.

Expression of the alcohol dehydrogenase gene, adhE, in Escherichia coli is anaerobically regulated at both the transcriptional and the translational levels. To study the AdhE protein, the adhE(+) structural gene was cloned into expression vectors under the control of the lacZ and trp(c) promoters. Wild-type AdhE protein produced under aerobic conditions from these constructs was inactive. Constitutive mutants (adhC) that produced high levels of AdhE under both aerobic and anaerobic conditions were previously isolated. When only the adhE structural gene from one of the adhC mutants was cloned into expression vectors, highly functional AdhE protein was isolated under both aerobic and anaerobic conditions. Sequence analysis revealed that the adhE gene from the adhC mutant contained two mutations resulting in two amino acid substitutions, Ala267Thr and Glu568Lys. Thus, adhC strains contain a promoter mutation and two mutations in the structural gene. The mutant structural gene from adhC strains was designated adhE*. Fragment exchange experiments revealed that the substitution responsible for aerobic expression in the adhE* clones is Glu568Lys. Genetic selection and site-directed mutagenesis experiments showed that virtually any amino acid substitution for Glu568 produced AdhE that was active under both aerobic and anaerobic conditions. These findings suggest that adhE expression is also regulated posttranslationally and that strict regulation of alcohol dehydrogenase activity in E. coli is physiologically significant.

Structure and function of the conserved 690 hairpin in Escherichia coli 16 S ribosomal RNA: analysis of the stem nucleotides.

Nucleotides 680 to 710 of Escherichia coli 16 S rRNA form a distinct structural domain required for ribosome function. The goal of this study was to determine the functional significance of pairing interactions in the 690 region. Two different secondary structures were proposed for this hairpin, based on phylogenetic and chemical modification studies. To study the effect of pairing interactions in the 690 hairpin on ribosome function and to determine which of the proposed secondary structures is biologically significant, we performed an instant-evolution experiment in which the nine nucleotides that form the proposed base-pairs and dangling ends of the 690 stem were randomly mutated, and functional mutant combinations were selected. A total of 96 unique functional mutants were isolated, assayed in vivo, and sequenced. Analysis of these data revealed extensive base-pairing and stacking interactions among the mutated nucleotides. Formation of either a Watson-Crick base-pair or G.U pair between positions 688 and 699 is absolutely required for ribosome function. We also performed NMR studies of a 31-nucleotide RNA which indicate the formation of a functionally important base-pair between nucleotides 688 and 699. Formation of a second base-pair between positions 689 and 698, however, is not essential for ribosome function, but the level of ribosome function correlates with the predicted thermodynamic stability of the nucleotide pairs in these positions. The universally conserved positions G690 and U697 are generally portrayed as forming a G.U mismatch. Our data show co-variation between these positions, but do not support the hypothesis that the G690:U697 pair forms a wobble structure. NMR studies of model 14-nt and 31-nt RNAs support these findings and show that G690 and U697 are involved in unusual stacking interactions but do not form a wobble pair. Preliminary NMR structural analysis reveals that the loop portion of the 690 hairpin folds into a highly structured and novel conformation.

Asymptomatic patients at high risk for deep venous thrombosis who receive inadequate prophylaxis should be screened.

BACKGROUND: Patients after stroke and major orthopedic surgery have increased factors for developing deep vein thrombosis. We sought to determine the implications of screening high-risk patients to detect proximal deep vein thrombosis. METHODS: We used decision analysis to determine the implications of screening vs not screening asymptomatic high risk patients with duplex ultrasonography to detect proximal deep venous thrombosis. The outcomes were bleeding, pulmonary embolism, death, and number of patients with true-positive, false-positive, and false-negative tests. RESULTS: Screening with ultrasonography, all asymptomatic patients who receive appropriate prophylaxis, prevalence 5%, would result in the treatment of 3.1% patients with proximal deep vein thrombosis (true positives); 2.9% without proximal deep vein thrombosis (false positives) and in the lack of diagnosis in 1.9% patients (false negatives). At a prevalence of 20%, no prophylaxis, screening would result in the treatment of 12.4% patients with proximal deep vein thrombosis (true positives), 2.4% without proximal deep vein thrombosis (false positives), and in the lack of diagnosis in 7.6% of patients (false negatives). CONCLUSIONS: Screening high-risk patients who receive prophylaxis is not warranted. Patients who receive no prophylaxis should be screened with ultrasonography.

Management outcomes in splenic injury: a statewide trauma center review.

OBJECTIVE: Clinical pathways now highlight both observation and operation as acceptable initial therapeutic options for the management of patients with splenic injury. The purpose of this study was to evaluate treatment trends for splenic injury in all North Carolina trauma centers over a 6-year period. METHODS: Splenic injuries in adults over a 6-year period (January 1988-December 1993) were identified in the North Carolina Trauma Registry using ICD-9-CM codes. Patients were divided into four groups by method of management: 1) no spleen operation, 2) splenectomy, 3) definitive splenorrhaphy, and 4) splenorrhaphy failure followed by splenectomy. The authors examined age, mechanism of injury, admitting blood pressure, and severity of injury by trauma score and injury severity score. SUMMARY BACKGROUND DATA: Comparisons were made between adult (17-64 years of age) and geriatric (older than 65 years of age) patients and between patients with blunt and penetrating injury. Resource utilization (length of stay, hospital charges) and outcome (mortality) were compared. RESULTS: One thousand two hundred fifty-five patients were identified with splenic injury. Rate of splenic preservation increased over time and was achieved in more than 50% of patients through nonoperative management (40%) and splenorrhaphy (12%). Splenorrhaphy was not used commonly in either blunt or penetrating injury. Overall mortality was 13%. Geriatric patients had a higher mortality and resource utilization regardless of their mechanism of injury or method of management. CONCLUSIONS: Nonoperative management represents the prevailing method of splenic preservation in both the adult and geriatric population in North Carolina trauma center hospitals. Satisfactory outcomes and economic advantages accompany nonoperative management in this adult population.

In vivo determination of RNA structure-function relationships: analysis of the 790 loop in ribosomal RNA.

The 790 loop is a conserved hairpin located between positions 786 and 796 of Escherichia coli 16 S rRNA that is required for ribosome function. Using a novel genetic approach, all positions in the loop were simultaneously mutated and functional mutant sequences were selected in vivo. This "instant evolution" experiment revealed that approximately 190 of the 262,144 possible mutant sequences were functional. Analysis of functional mutant sequences allowed discrimination between nucleotides directly involved in protein synthesis and those involved primarily in loop structure. Among the functional mutant sequences, positions 789 and 791 were invariant and extensive covariation was observed among the nucleotides at the base of the loop at positions 787, 788, 794 and 795. NMR and thermodynamic analyses of model 790 hairpins in vitro revealed weak pairing interactions between positions 787 and 795 and between positions 788 and 794 consistent with the in vivo mutational analysis. Functional analysis of site-directed mutants containing all possible nucleotide combinations at positions 787 and 795 in vivo showed that stable base-pairs at these positions prevent subunit association.

Quantitative densitometry of proteins stained with coomassie blue using a Hewlett Packard scanjet scanner and Scanplot software.

In the present study we evaluated the performance of a software/scanner system that employed the Hewlett Packard (HP) ScanJet Plus and Scanplot Software for densitometric quantification of protein loads stained with Coomassie Brilliant Blue following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Gels with bovine serum albumin (BSA) standards, ranging from 0.125 to 10 micrograms, were scanned using reflectance densitometry with 127 microns step size in both the x and y directions and a resolution of 200 dots per inch. Densitometric volume was calculated for each protein band from scanner output in the tagged image file format (TIFF) by a customized software package, Scanplot V. 4.05 (Cunningham Engineering). Protein loads between 0.125 and 10.0 micrograms vs. volume were fit by a second-order regression: Volume = -0.58 x protein load2 + 16.82 x protein load + 7.87 (r = 0.991, p < 0.01). The same gels were scanned and quantified using a transmittance laser densitometer; densitometric volumes measured by both systems were highly correlated (r2 = 0.981, p < 0.01). Additional gels of BSA, smooth muscle myosin heavy chain (myosin), and actin displayed linear relationships between protein loads up to 4.0 micrograms and densitometric volume reflecting unique dye binding properties. We conclude that accurate and reproducible quantitative densitometry of SDS-PAGE can be performed using the HP ScanJet Plus scanner and Scanplot software.

Genetic analysis of the Shine-Dalgarno interaction: selection of alternative functional mRNA-rRNA combinations.

The interaction of bacterial mRNAs with the small ribosomal subunit is strongly promoted by Watson-Crick base pairing between a purine-rich consensus ribosomal RNA-binding sequence (RBS) on mRNA and its complementary message-binding sequence (MBS) on rRNA known as the Shine-Dalgarno interaction. To identify and characterize components of the Shine-Dalgarno interaction that contribute to translation initiation, we simultaneously and randomly mutated both the MBS of the 16S rRNA gene from Escherichia coli and the RBS of the chloramphenicol acetyl transferase (CAT) gene and selected chloramphenicol-resistant mutant combinations. Nucleotide distribution in both mutated sequences of the survivors was nonrandom and the MBSs of the surviving clones showed a preference for purines. In addition, strong interactions between specific nucleotide pairs within each of the mutated sequences were indicated. Although the contribution of free energy of duplex formation between rRNA and mRNA was highly significant (P < 0.001), only 23% of the observed activity in all of the mutants could be attributed to this variable. MBSs that were lethal upon expression were also isolated. These sequences may cause overtranslation of specific messages in the cell. These data indicate that specific sequence constraints exist (primarily within the MBS) that are necessary to establish a functional threshold for translation and that only after establishment of this threshold is the level of expression significantly affected by the free energy of MBS-RBS duplex formation.

The ripped cava.

Lacerations of the inferior vena cava are associated with a high mortality and may be difficult to repair. The majority of injuries are due to penetrating trauma. Rapid transportation to definitive surgical care with effective resuscitation may improve mortality. Surgical management includes adequate treatment of hypovolemic shock due to blood loss. Placement of intravenous infusion sites below the level of the diaphragm may be effective. Operative control of the inferior vena cava can be accomplished by directed digital compression followed by a proximal and distal control. Injuries of the inferior vena cava above the level of the renal veins are associated with an increased mortality. Retrohepatic and subdiaphragmatic injuries are highly lethal. This article discusses appropriate surgical approaches for repair of the inferior vena cava above and below the diaphragm.

Methylation of the conserved A1518-A1519 in Escherichia coli 16S ribosomal RNA by the ksgA methyltransferase is influenced by methylations around the similarly conserved U1512.G1523 base pair in the 3' terminal hairpin.

An in vitro system developed for the site-specific mutagenesis of 16S rRNA of Escherichia coli ribosomes was used to make five mutations around the highly conserved U1512.G1523 base pair in the 3' terminal hairpin. Each of the mutant RNAs was reconstituted with a complete mixture of 30S proteins to yield 30S ribosomal particles, which were tested for the ability of the ksgA methylase to form m6(2)A1518 and m6(2)A1519. Dimethylation of A1518 and A1519 in the hairpin loop was inhibited 20-80% by the mutations. The results indicate that G1523 and C1524 in the stem are important determinants for the dimethylation of A1518 and A1519 in the loop. Either the enzyme recognition region extends that far or the effect of mutations in the stem are propagated in some manner to the loop. The conserved U.G base pair does not of itself appear to play a major role in ksgA methylase recognition.

Intravenous ethanol for alcohol detoxification in trauma patients.

Traumatic injury frequently follows alcohol abuse. Between October 1, 1988 and January 31, 1992, 2,219 patients were admitted to the Trauma Service at the University Medical Center of Eastern Carolina-Pitt County. Of the 1,602 who were tested for serum ethanol, 685 (43%) were found to have measurable levels. Thirty-seven patients had alcohol withdrawal and were treated with intravenous ethanol; 34 were male (21 black, 13 white) and 3 female (1 black, 2 white), with an average age of 46 years. Those who had withdrawal had an average serum ethanol level, on presentation, of 239 mg/dL (N = 34). Fourteen patients were involved in motor vehicle crashes, seven were pedestrians struck by cars, and the remaining 16 had various traumatic mechanisms of injury. The most common injuries were long-bone fractures and blunt abdominal trauma. The length of ethanol therapy averaged 4 days. A majority of patients had a favorable response to treatment. Relative contraindications to i.v. ethanol therapy were CNS trauma, liver disease, and pancreatitis. i.v. ethanol is a safe and effective method of alcohol detoxification in the trauma patient.

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., Bakin, A., Weitzmann, C. J., Pflumm, M., & Ofengand, J. (1992) Biochemistry 31, 12012-12022]. Here we show that the additional nearby tertiary base pairs C1404:G1497 and G1405:C1496 also exist and are functionally important for tRNA binding to the ribosomal A and P sites. Breakage of the base pairs in turn led to a loss of activity at both A and P sites, whereas restoration in the reverse orientation led to recovery of activity. Recovery was incomplete, indicating that base pairing alone is insufficient for full restoration of function. Mutation of U1498 to G created the potential for the tertiary base pair C1403:G1498, which could stack on the aforementioned double base pair, creating a more stable helix longer by one residue. This mutation did not affect subunit association, A- and P-site binding of tRNA to 70S, fMet-tRNA binding to 30S, or poly(Phe) synthesis but did block formation of the first peptide bond, fMet-Val. Mutation of U1498 to A or C did not show this effect. Since the G1498 mutant could make both the 70S initiation complex and the peptide bond, as shown by its ability to form fMet-puromycin, the block in fMet-Val synthesis appears to involve some aspect of A-site function.(ABSTRACT TRUNCATED AT 250 WORDS)