Biomedical subjects

Piotr Mucha

Publications and source records attributed to Piotr Mucha.

5 recordsLinked to original sources

PubMed2004

Sequence-altered peptide adopts optimum conformation for modification-dependent binding of the yeast tRNAPhe anticodon domain.

Amino acid contributions to protein recognition of naturally modified RNAs are not understood. Circular dichroism spectra and predictive software suggested that peptide tF2 (S1ISPW5GFSGL10 LRWSY15), selected from a phage display library to bind the modified anticodon domain of yeast tRNAPhe (ASL), adopted a beta-sheet structure. Ala residues incorporated at positions Pro4 and Gly6, both predicted to be involved in a turn, did not alter the peptide binding affinity for the ASLPhe, although major changes in the peptide's CD spectra were observed. Substitutions at three positions Pro4, Gly6, and Gly9, the latter not predicted to be in a turn, reduced the peptide's binding affinity to 4% of that of the unsubstituted tF2 and strongly influenced the peptide's secondary structure. The results suggest that peptides with different conformations, but similar affinities, adopt the optimal binding conformation, indicative of a structurally adaptive model of binding in which the modified RNA serves as a scaffold.

Anticodon↗

PubMed2003

Using capillary electrophoresis to study methylation effect on RNA-peptide interaction.

Methylation of RNA and proteins is one of a broad spectrum of post-transcriptional/translational mechanisms of gene expression regulation. Its functional signification is only beginning to be understood. A sensitive capillary electrophoresis mobility shift assay (CEMSA) for qualitative study of the methylation effect on biomolecules interaction is presented. Two RNA-peptide systems were chosen for the study. The first one consists of a 17-nucleotide analogue (+27-+43) of the yeast tRNA(Phe) anticodon stem and loop domain (ASL(Phe)) containing three of the five naturally occurring modifications (2'-O-methylcytidine (Cm(32)), 2'-O-methylguanine (Gm(34)) and 5-methylcytidine (m(5)C(40))) (ASL(Phe)-Cm(32),Gm(34),m(5)C(40)) and a 15-amino-acid peptide (named t(F)2: Ser(1)-Ile-Ser-Pro-Trp(5)-Gly-Phe-Ser-Gly-Leu(10)-Leu- Arg-Trp-Ser-Tyr(15)) selected from a random phage display library (RPL). A peptide-concentration-dependent formation of an RNA-peptide complex was clearly observable by CEMSA. In the presence of the peptide the capillary electrophoresis (CE) peak for triply methylated ASL(Phe) shifted from 18.16 to 20.90 min. Formation of the complex was not observed when an unmethylated version of ASL(Phe) was used. The second system studied consisted of the (+18)-(+44) fragment of the trans-activation response element of human immunodeficiency virus type 1 (TAR RNA HIV-1) and a 9-amino-acid peptide of the trans-activator of transcription protein (Tat HIV-1) Tat(49-57)-NH(2) (named Tat1: Arg(49)-Lys-Lys-Arg(52)-Arg-Gln-Arg-Arg- Arg(57)-NH(2)). In the presence of Tat(49-57)-NH(2) a significant shift of migration time of TAR from 18.66 min to 20.12 min was observed. Methylation of a residue Arg(52)-->Arg(Me)(2), crucial for TAR binding, strongly disrupted formation of the complex. Only at a high micromolar peptide concentration a poorly shaped, broad peak of the complex was observed. CE was found to be an efficient and sensitive method for the analysis of methylation effects on interaction of biomolecules.

Amino Acid Sequence↗

PubMed2002

Structural requirements for conserved Arg52 residue for interaction of the human immunodeficiency virus type 1 trans-activation responsive element with trans-activator of transcription protein (49-57). Capillary electrophoresis mobility shift assay.

A sensitive capillary electrophoresis mobility shift assay (CEMSA) for qualitative study of the interaction between the trans-activation response element (TAR) and the trans-activator of transcription protein (Tat) has been presented. The human immunodeficiency virus type 1 (HIV-1) Tat promotes elongation of viral mRNAs binding to the TAR. It has been suggested that a single, conserved arginine residue (presumably Arg52) within the arginine-rich region (ARR) of Tat plays the major role for the Tat-TAR recognition. To study structural requirements of the Arg52 position, Tat(49-57)-NH2 analogues substituted with nonencoded amino acids at the Arg52 position have been synthesized and their interaction with TAR has been studied by CEMSA. Using a linear polyacrylamide-coated capillary and a sieving polymer containing separation buffer, well separated and shaped peaks of free and bound TAR RNA were obtained. In the presence of Tat1 peptide bearing the native sequence of Tat(49-57) a significant shift of migration time of TAR from 18.66 min (RSD=1.4%) to 20.12 min (RSD=2.4%) was observed. We have found that almost every substitution within the guanidino group of the Arg52 [L-Arg52-->Cit, -->Orn, -->Arg(NO2), -->Arg(Me2)] strongly disrupted or abolished the TAR-Tat peptide interaction. Enantiomeric substitution, L-Arg52-->D-Arg was the only one which notably promoted TAR-Tat peptide interaction. The results demonstrate that the specific net of hydrogen bonds created by the guanidinio group of conserved Arg52 plays a crucial role for TAR-Tat HIV-1 recognition. The newly developed procedure describes for the first time use of CE to monitor RNA-peptide complex formation. The methodology presented should be generally applicable to study RNA-peptide (protein) interaction.

Amino Acid Sequence↗

PubMed2002

Interaction of RNA with phage display selected peptides analyzed by capillary electrophoresis mobility shift assay.

A sensitive capillary electrophoresis mobility shift assay (CEMSA) to analyze RNA/peptide interactions has been developed. Capillary electrophoresis (CE) has been adapted for investigating the interaction between variously methylated 17-nt analogs of the yeast tRNAPhe anticodon stem and loop domain (ASL(Phe)) and 15-amino-acid peptides selected from a random phage display library (RPL). A peptide-concentration-dependent formation of RNA/peptide complex was clearly visible during CEMSA. In the presence of peptide, the UV-monitored CE peak for ASLPhe with three of the five naturally occurring modifications (2'-O-methylcytidine (Cm32), 2'-O-methylguanine (Gm34) and 5-methylcytidine (m5C40) shifted from 18.16 to 20.90 min. The mobility shift was observed only for methylated RNA. The negative effects of diffusion, electroosmotic flow and adhesion of molecules to the capillary internal wall were suppressed by using a buffer containing a sieving polymer and a polyacrylamide-coated capillary. Under these conditions, well-shaped peaks and resolution of RNA free and bound to peptide were achieved. Peptide tF2, the most populated ligand in the RPL, specifically bound triply methylated ASLPhe in a methylated nucleoside-dependent manner. CE was found to be an efficient and sensitive method for the qualitative analysis of RNA-peptide interaction and should be generally applicable to the study of RNA-peptide (protein) interactions.

Amino Acid Sequence↗

PubMed2002

Aminoacyl-tRNA synthetases and aminoacylation of tRNA in the nucleus.

This review is focused on findings concerning the presence of translation apparatus components (aminoacyl-tRNA synthetases, aminoacyl-tRNA, elongation factors) as well as translation itself in the nucleus. A nuclear role of these molecules is unknown. New findings suggest that well-accepted model of spatial segregation of transcription and translation in eukaryotic cell may be oversimplifcation. Nuclear coupling of both these processes show us how exciting and surprising may be the world of the living cell.

Active Transport, Cell Nucleus↗