Biomedical subjects

T Tsalkova

Publications and source records attributed to T Tsalkova.

9 recordsLinked to original sources

PubMed1999

The effect of a hydrophobic N-terminal probe on translational pausing of chloramphenicol acetyl transferase and rhodanese.

The effect on translational pausing of a hydrophobic probe, coumarin, at the N terminus of nascent peptides was investigated. Two different proteins, bacterial chloramphenicol acetyltransferase and bovine rhodanese, were synthesized by coupled transcription/translation in a cell-free system derived from Escherichia coli. Protein synthesis was initiated with N-formyl-Met-tRNAf or N-acetyl-S-coumarin-Met-tRNAf. Cotranslational incorporation of the coumarin derivative generated nascent polypeptides with a hydrophobic residue at their N termini. The effect of the two N-terminal groups on the size distribution and quantity of the peptides formed by translational pausing was investigated. The N-terminal coumarin caused an accumulation of nascent chloramphenicol acetyltransferase peptides in the mass range of 3.5-4.0 kDa that reflects a delay in translation at this point. No similar effect on rhodanese pause-site peptides was observed. This effect on translational pausing cannot be explained by either mRNA secondary structure or rare codons and tRNA abundance. It is suggested that the effect of N-terminal coumarin on translational pausing is the result of the interaction of the nascent peptide with components of the large ribosomal subunit along the path it follows between the peptidyl transferase center and the exit site on the distal surface.

Animals↗

PubMed1999

Co-translational folding.

Nascent proteins appear to fold co-translationally. The ribosome itself may function as a chaperone, providing a sheltered environment in which the nascent peptide is protected from aggregation and degradation, and in which folding into the tertiary structure is facilitated by interactions both with ribosomal proteins and with specific segments of the ribosomal RNA.

Amino Acid Sequence↗

PubMed1999

N-terminal and C-terminal modifications affect folding, release from the ribosomes and stability of in vitro synthesized proteins.

Important aspects of translation are release and folding of the synthesized protein into its three-dimensional structure. Studies from our group indicated that during in vitro protein synthesis a large portion of full-length polypeptides apparently accumulated as peptidyl-tRNA on ribosomes. We have also shown that some proteins though released in biologically active form may be inactivated without being degraded. These experiments were carried out by coupled transcription/translation using an Escherichia coli extract in which eukaryotic or prokaryotic test proteins were synthesized from their coding sequence inserted into specific plasmids. Experiments described here were designed to analyze the effects of N-terminal and C-terminal modifications of the coding sequence on the ribosomal release/termination process and on the stability of the newly synthesized protein. Elimination of the leader sequence in two proteins tested, mitichondrial rhodanese and bacterial beta-lactamase, caused an increase in the percentage of polypeptides released from the ribosomes relative to total synthesis. Conversely, an N-terminal extension such as a histidine-lag impaired the ribosomal release process. Also, a hydrophobic N-terminal modification of the synthesized protein reduced release of newly formed protein from the ribosomes. A C-terminal extension of the coding sequence for rhodanese by one amino acid decreased the percentage released polypeptide and furthermore affected the stability of the in vitro formed protein. We propose that a regulatory mechanism exists by which N-terminal and C-terminal sequences of a newly synthesized protein have feed-back effects on the termination factor-mediated release and on the stability of the native three-dimensional structure.

Cell-Free System↗

PubMed1998

Truncations at the NH2 terminus of rhodanese destabilize the enzyme and decrease its heterologous expression.

Rhodanese mutants containing sequential NH2-terminal deletions were constructed to test the distinct contributions of this region of the protein to expression, folding, and stability. The results indicate that the first 11 residues are nonessential for folding to the active conformation, but they are necessary for attaining an active, stable structure when expressed in Escherichia coli. Rhodanese species with up to 9 residues deleted were expressed and purified. Kinetic parameters for the mutants were similar to those of the full-length enzyme. Compared with shorter truncations, mutants missing 7 or 9 residues were (a) increasingly inactivated by urea denaturation, (b) more susceptible to inactivation by dithiothreitol, (c) less able to be reactivated, and (d) less rapidly inactivated by incubation at 37 degreesC. Immunoprecipitation showed that mutants lacking 10-23 NH2-terminal amino acids were expressed as inactive species of the expected size but were rapidly eliminated. Cell-free transcription/translation at 37 degreesC showed mutants deleted through residue 9 were enzymatically active, but they were inactive when deleted further, just as in vivo. However, at 30 degreesC in vitro, both Delta1-10 and Delta1-11 showed considerable activity. Truncations in the NH2 terminus affect the chemical stability of the distantly located active site. Residues Ser-11 through Gly-22, which form the NH2-proximal alpha-helix, contribute to folding to an active conformation, to resisting degradation during heterologous expression, and to chemical stability in vitro.

Amino Acid Sequence↗

PubMed1998

Different conformations of nascent peptides on ribosomes.

The length at which the N terminus of nascent proteins becomes available to antibodies during their synthesis on ribosomes was determined. Three different proteins, bovine rhodanese, bacterial chloramphenicol acetyltransferase and MS2 coat protein, were synthesized with coumarin at their N terminus in a cell-free system derived from Escherichia coli. A derivative of coumarin was cotranslationally incorporated as N-coumarin-methionine at the N terminus of polypeptides. The interaction of specific anti-coumarin antibodies with this N-terminal coumarin of ribosome-bound nascent peptides was examined. The results indicate that short nascent peptides of each of the three proteins are unreactive, that the length at which they become accessible to the antibodies is different for the three proteins, and that longer peptides differ in their reactivity. It is suggested that these differences are due to differences in the conformation acquired by the peptides as they are synthesized on the ribosomes.

Capsid↗

PubMed1993

GroEL and GroES increase the specific enzymatic activity of newly-synthesized rhodanese if present during in vitro transcription/translation.

Enzymatically active mammalian rhodanese, a mitochondrial matrix enzyme, which has been found to require assistants for efficient refolding in vitro, has been synthesized from a plasmid in a cell-free, fractionated, coupled transcription/translation system derived from Escherichia coli. The bacterial chaperonins, GroEL and GroES, along with the rhodanese substrate thiosulfate greatly enhance the specific enzymatic activity of the rhodanese polypeptide that is formed. Indirect evidence suggests that the effect of the GroEL/ES chaperonins is on ribosome-bound nascent peptides. The in vitro transcription/translation system produces sufficient amounts of rhodanese to provide a system for studying factors that control the initial steps in folding of nascent proteins.

Bacterial Proteins↗

PubMed1992

Structural dynamics of translating ribosomes.

We describe three groups of small angle neutron scattering (SANS) experiments with translating ribosomes: 1) regular protonated (normal abundance hydrogen) particles; 2) two isotopic hybrid particles which are reconstituted from one protonated and the other deuterated subunit; 3) four isotypic hybrid particles differing from each other by the extent of protein and RNA deuteration. Using the SANS contrast variation method the radii of gyration of protein and RNA components in both ribosomal subunits as well as the intersubunit distance in the pre- and post-translocation states were determined. The results obtained suggest the following model of the ribosome as a dynamic machine. The ribosome oscillates between two major conformers differing in geometrical dimensions. The 'active' (pulsating) part of the ribosome is the 30S subunit. We believe that the movement of its 'head' relative to the passive 50S subunit is the main mechanical act of translocation. The radius of gyration of the 30S subunit and the intersubunit distance change upon the movement. This is corroborated by neutron scattering data.

Bacterial Proteins↗

PubMed1991

The synthesis of polyphenylalanine on ribosomes to which erythromycin is bound.

Erythromycin binds to the large subunit of Escherichia coli ribosomes at a specific site that is very close to the amino acid of aminoacyl-tRNA bound into the peptidyltransferase center, and to the site to which puromycin is bound, the P and A sites, respectively, of the classical two-site model of ribosome function. Both erythromycin and puromycin affect fluorescence from fluorescent derivatives of aminoacyl-tRNAs, while both puromycin and aminoacyl-tRNAs affect fluorescence of fluorescent derivatives of erythromycylamine. The results demonstrate unequivocally that erythromycin, deacylated tRNA, a peptidyl-tRNA analogue and puromycin can be bound simultaneously to the same ribosome. Nascent peptides of more than a few amino acids in length block binding of erythromycin to the ribosomes but, unlike most other peptides, long polyphenylalanine chains can be synthesized on ribosomes to which erythromycin is bound. It is suggested that this refractory synthesis in the presence of erythromycin reflects the atypical physical and structural properties of polyphenylalanine.

Binding Sites↗

PubMed1991

The conformation of nascent polylysine and polyphenylalanine peptides on ribosomes.

Polypeptide synthesis using either phenylalanine or lysine was initiated on Escherichia coli ribosomes; then the position and conformation of the nascent peptide were monitored by fluorescence techniques. To this end, fluorophores had been attached to the amino terminus of each nascent peptide, and major differences were observed as chain extension occurred. Polyphenylalanine appeared to build up as a hydrophobic mass adjacent to the peptidyl transferase center while polylysine apparently was extended directly from the ribosome into the surrounding solution. An explanation for these differences may be provided by the physical and chemical properties of each polypeptide. These properties may be responsible for the route by which each peptide exits the peptidyl transferase center as demonstrated by the different sensitivity of each to inhibition by erythromycin.

Dipeptides↗