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C M Spahn

Publications and source records attributed to C M Spahn.

30 records · Page 2Linked to original sources
PubMed1998

Mutational analysis of the donor substrate binding site of the ribosomal peptidyltransferase center.

Previous experiments have shown that the top of helix 90 of 23S rRNA is highly important for the ribosomal peptidyltransferase activity and might be part of the donor (P) site. Developing on these studies, mutations in the 23S rRNA at the highly conserved positions G2505, G2582, and G2583 were investigated. None of the mutations affected assembly, subunit association, or the capacity of tRNA binding to A and P sites. A "selective transpeptidation assay" revealed that the mutations specifically impaired peptide bond formation. Results with a modified "fragment" assay using the minimal donor substrate pA-fMet are consistent with a model where the nucleotides psiGG2582 form a binding pocket for C75 of the tRNA.

Adenosine Monophosphate↗
PubMed1996

Mutational analysis of two highly conserved UGG sequences of 23 S rRNA from Escherichia coli.

The 23 S-type rRNA contains two phylogenetically conserved UGG sequences, which have the potential to bind the universal CCA-3'-ends of tRNAs at the ribosomal peptidyltransferase center by base pairing. The first two positions, UG, of these sequences at the helix-loop 80 (U2249G2250) and helix-loop 90 (Psi2580G2581) and some related nucleotides were tested by site-directed mutagenesis for their involvement in ribosomal function, i.e. peptidyltransferase. The plasmid-derived mutated 23 S rRNA comprised about 50% of the total 23 S rRNA. None of the single mutations caused an assembly defect, and all 50 S subunits carrying an altered 23 S rRNA could freely exchange with the pools of 70S ribosomes and polysomes. The mutations at the helix-loop 80 region hardly affected bacterial growth. However, mutations at the helix 90 caused severe growth effects and severely impaired the in vitro protein synthesis, showing that this 23 S rRNA region is of high importance for ribosomal function.

Base Sequence↗
PubMed1996

Conserved nucleotides of 23 S rRNA located at the ribosomal peptidyltransferase center.

Two nucleotides of the 23 S rRNA gene were mutated; the nucleotides correspond to the first two positions of the universally conserved sequence PsiGG2582 at the peptidyltransferase ring of 23 S rRNA. The ribosomes containing the altered 23 S rRNA were analyzed. Previously, it was shown that ribosomal assembly was indistinguishable from that in wild-type cells, that the flow of the corresponding 50 S subunit into the polysome fraction was not restricted, but that the ribosomes were strongly impaired in poly(Phe) synthesis (C. M. T. Spahn, J. Remme, M. A. Schäfer, and K. H. Nierhaus (1996) J. Biol. Chem. 271, 32849-32856). Here we apply assay systems exclusively testing the puromycin reaction of ribosomes carrying plasmid-born rRNA, a dipeptide assay using the minimal P site donor pA(fMet) and a translocation system not depending on the puromycin reaction. The mutations in helix 90 exclusively abolish or severely impair the ribosome capability to catalyze AcPhe-puromycin formation. A possible explanation of these observations is that G2581 and Psi2580 (and possibly also G2582) are part of the binding site of C75 of peptidyl-tRNA in the P site. The results suggest that in this case, however, such an interaction would disobey canonical base pairing.

Base Sequence↗
PubMed1996

Effects of antisense DNA against the alpha-sarcin stem-loop structure of the ribosomal 23S rRNA.

Antisense DNAs complementary against various sequences of the alpha-sarcin domain (C2646-G2674) of 23S rRNA from Escherichia coli were hybridized to naked 23S rRNA as well as to 70S ribosomes. Saturation levels of up to 0.4 per 70S ribosome were found, the identical fraction was susceptible to the attack of the RNase alpha-sarcin. The hybridization was specific as demonstrated with RNase H digestion, sequencing the resulting fragments and blockage of the action of alpha-sarcin. The RNase alpha-sarcin seems to approach its cleavage site from the 3' half of the loop of the alpha-sarcin domain. Hybridization is efficiently achieved at 37 degrees C and can extend at least into the 3' strand of the stem of the alpha-sarcin domain. However, the inhibition of alpha-sarcin activity is observed at 30 degrees C but not at 37 degrees C. For a significant inhibition of poly(Phe) synthesis the temperature had to be lowered to 25 degrees C. The results imply that the alpha-sarcin domain changes its conformation during protein synthesis and that the conformational changes may include a melting of the stem of the alpha-sarcin domain.

Base Sequence↗
PubMed1996

Throwing a spanner in the works: antibiotics and the translation apparatus.

The protein synthetic machinery is essential to all living cells and is one of the major targets for antibiotics. Knowledge of the structure and function of the ribosome and its associated factors is key to understanding the mechanism of drug action. Conversely, drugs have been used as tools to probe the translation cycle, thus providing a means to further our understanding of the steps that lead to protein synthesis. Our current understanding as to how antibiotics disrupt this process is reviewed here, with particular emphasis on the prokaryotic elongation cycle and those drugs that interact with ribosomal RNAs.

Anti-Bacterial Agents↗
PubMed1996

[Phosphate sites of RNA-ligands interacting with ribosome at different stages of translation. Thiophosphate method of analysis].

A novel footprinting method was recently developed which identifies phosphate groups of RNA involved in strong RNA-RNA and RNA-protein interactions. The method is based on iodine-dependent RNA cleavage at phosphothioate groups as long as these groups are not protected from iodine. Our recent studies of mRNA and tRNA regions protected in active ribosomes are summarized; initiation state of ribosomes as well as two elongation states in pre- and post-translocational states were analyzed. Only one phosphate group of mRNA, which was two positions upstream of the decoding codons, was weakly protected in longation complexes, whereas this group and the phosphate groups in the Shine-Dalgarno sequence were protected in the initiation complex. No protection was observed downstream of the decoding codons. On the contrary, numerous phosphate residues of tRNA were protected by the ribosome. The tRNA protection patterns significantly varied between two tRNAs simultaneously bound to the ribosome. The protection pattern of an individual tRNA was not significantly affected by translocation. The data indicate that both tRNA molecules are tightly bound to the ribosome, whereas mRNA is fixed predominantly by two tRNAs via codon-anticodon interaction. A possible translocation mechanism is suggested.

Base Sequence↗
PubMed1995

Interaction of tRNAs with the ribosome at the A and P sites.

In vitro transcribed tRNA(Phe) analogues from Escherichia coli containing up to four randomly distributed A, G, U or C phosphorothioated nucleotides were used to investigate contact patterns with the ribosome in the A and P sites. The tRNAs were biologically active. Molecular iodine (I2) can trigger a break in the sugar-phosphate backbone at phosphorothioated positions of the ribosomal bound tRNAs if contacts with ribosomal components do not prevent access of the iodine. Highly differentiated protection patterns were found which were strikingly different in the A and P sites, respectively. Strong protections accumulated in the T psi C loop and no protection was seen in the extra-arm region in both sites, whereas the phosphates in the anticodon loop are more strongly protected in the A site. Strong common protections in both the A and P sites were found neighbouring universally or semi-universally conserved bases in prominent regions of the tertiary structure of tRNAs: Y11, Y32, U33, psi55, C56, A58 and Y60. These bases are therefore candidates for 'identity elements' in ribosomal tRNA recognition. The data further indicate that tRNAs change their conformations upon binding to either ribosomal site.

Acetylation↗
PubMed1990

Similarities and differences in the inhibition patterns of thiostrepton and viomycin: evidence for two functionally different populations of P sites when occupied with AcPhe-tRNA.

According to the allosteric three-site model for the ribosomal elongation cycle, the reactions from the pre- to the post-translocational state and vice versa represent allosteric transitions which are catalyzed by elongation factor (EF)-G and EF-Tu, respectively. It has been shown recently that the non-related antibiotics thiostrepton and viomycin inhibit protein biosynthesis via a surprisingly similar mechanism. Both drugs primarily block the allosteric transitions in either direction (Hausner et al. (1988) J. Biol. Chem. 263, 13103-13111). Here we show that the secondary effects of these antibiotics differ strikingly. When the P site of poly(U) programmed ribosomes is quantitatively filled with AcPhe-tRNA, thiostrepton stimulates the rate of the formation of AcPhe-puromycin 2-fold, whereas viomycin inhibits the puromycin reaction (up to 75% inhibition). The thiostrepton-dependent stimulation is only observed when the drug is given before the P site is occupied; when thiostrepton is added after pre-filling the P site, the peptidyltransferase activity is not affected, in contrast to the translocation reaction, which is blocked irrespective of whether the drug is administered before or after tRNA is bound. The effects of both drugs became distinctly more pronounced when the P sites were saturated with AcPhe-tRNA as compared to half-saturated ribosomes. We conclude that roughly one half of the ribosomes, which first bind AcPhe-tRNA to the P site, carry this ligand in a different orientation to that of the second half of the ribosome population. These two populations probably reflect the P site in the pre- and post-translocational state, respectively.

Allosteric Regulation↗
PubMedDate not supplied

Prediction of oxygen uptake and energy expenditure during exercise in obese women.

PURPOSE: For patients concerned with weight loss, monitoring the energy balance between daily dietary intake and exercise expenditure can be useful. Formulas commonly used to estimate the energy costs of exercise were previously derived from healthy men of normal body weight. The purpose of this study was to determine the relationship between measured and predicted exercise energy expenditure for obese women. METHODS: Oxygen uptake (VO2) was measured using respiratory gas analysis in 45 obese (92 +/- 16 kg; 40 +/- 7% fat) and 10 normal weight (control) (58 +/- 5 kg; 21 +/- 6% fat) women during progressive exercise on a motorized treadmill. VO2 was also calculated at matched workrates using a regression equation published by the American College of Sports Medicine. The relationship between predicted versus measured VO2 was determined using least squares regression analysis. RESULTS: The slope of the regression line for measured versus predicted VO2 for controls (y = 0.98x +/- 0.56; P < .001) was different than that of obese women (y = 0.75x +/- 3.06; P < .001). The slope of the regression line for controls was in close approximation to the line of identity, whereas the slope for obese was below it. Using VO2 to calculate kcal, measured energy expenditure, was significantly lower than predicted energy expenditure for obese subjects, but not for controls at several matched workrates: Stage III (213 +/- 40 versus 225 +/- 38 kcal per 30 minutes, P < .001); stage 4 (292 +/- 55 versus 340 +/- 58 kcal per 30 minutes, P < .001); and stage 5 (330 +/- 55 versus 412 +/- 70 kcal per 30 minutes, P < .001) obese measured versus obese predicted, respectively. CONCLUSIONS: The authors conclude that the standard prediction equation gives a better estimation of VO2 for women who have average body weight and body fat than for obese women. This may, in part, be due to the differences in weight and/or fat mass between these subjects and those used to derive this equation. These findings should be considered when estimates of VO2 and energy expenditure are used rather than direct measures for obese women.

Adult↗
PubMedDate not supplied

Clinical profile and outcomes of obese patients in cardiac rehabilitation stratified according to National Heart, Lung, and Blood Institute criteria.

PURPOSE: Obesity is a major health problem and must be evaluated and treated in cardiac rehabilitation patients. The purpose of this study was to identify the scope of this problem in an urban-based cardiac rehabilitation program by evaluating the prevalence of obesity, and comparing the clinical and risk factor profiles and outcomes of patients stratified according to National Heart, Lung, and Blood Institute (NHLBI) weight classifications. METHODS: Four hundred forty-nine consecutive cardiac rehabilitation patients, aged 57 +/- 11 years, were stratified according to the NHLBI criteria as: normal (body mass index [BMI] 18-24.9 kg/m2), overweight (BMI 25-29.9 kg/m2), class I/II obese (BMI 30-39.9 kg/m2), and class III morbidly obese (BMI > or = 40 kg/m2). Baseline cardiac risk factors and dietary habits were identified, and both pre- and postexercise training measurements of exercise tolerance, weight, and lipid profile were obtained. RESULTS: Overweight and obesity (BMI > or = 25 kg/m2) were present in 88% of patients. Compared to normal weight patients, obese patients were younger and had a greater adverse risk profile (higher prevalence of diabetes and hypertension, larger waist circumference, lower exercise capacity, lower high-density lipoprotein cholesterol level) at entry. After 10 weeks, all groups had a significant increase in exercise capacity, and on average obese patients in each category lost weight (Class I/II--4 lbs and Class III--12 lbs). Dropout rates were similar among the groups. CONCLUSION: Overweight and obesity are highly prevalent in cardiac rehabilitation. Overweight and obese patients had a greater adverse cardiovascular risk profile, including a lower exercise capacity in the latter. Thus, targeted interventions toward weight management in contemporary cardiac rehabilitation programs are important. Although short-term outcomes appear promising, greater efforts to improve these outcomes and to support long-term management are needed.

Aged↗
PubMedDate not supplied

The elongating ribosome: structural and functional aspects.

We determined the positions and arrangements of RNA ligands within the ribosome with a new neutron-scattering technique, the proton-spin contrast-variation. Two tRNAs were bound to the ribosome in the pre-translocational and the post-translocational state. The mass centre of gravity of both tRNAs resides at the subunit interface of the body of the 30S subunit. Both tRNAs are separated by an angle of 50-55 degrees, and their mutual arrangement does not change during translocation. The mass centre of gravity moves by 13 +/- 3 A (1A = 0.1 nm) during translocation, corresponding well with the length of one codon. Using an RNase-digestion technique, the length of the mRNA sequence covered by the ribosome was determined to be 39 +/- 3 nucleotides before and after translocation. The ribosome moves like a rigid frame along the mRNA during translocation. In contrast, both tRNAs seem to be located on a movable ribosomal domain, which carries the tRNAs before, during, and after translocation, leaving the microtopography of the tRNAs with the ribosome unaltered. This conclusion was derived from an analysis of the contract patterns of thioated tRNAs on the ribosome. The results have led to a new model of the elongation cycle, which reinterprets the features of the previous "allosteric three-sites model" in a surprisingly simple fashion. Finally, a mutational analysis has identified a single nucleotide of the 23S rRNA essential for the peptidyltransferase activity.

Amino Acid Sequence↗