Cotranslational folding--omnia mea mecum porto?

Evidence for cotranslational folding on both prokaryotic and eukaryotic ribosomes is reviewed. Molecular chaperones appear to assist only a small fraction of newly synthesized proteins in folding into their native conformation. The recently published crystal structure of the large ribosomal subunit at 2.5 A resolution has provided the basis for understanding where and how peptide synthesis takes place on the ribosome. The nascent peptide is concluded to pass through a tunnel that extends about 100 A between the peptidyl transferase center and its exit site. The minimum diameter of the tunnel and the apparent physical and chemical properties of its walls appear to preclude complex folding of the nascent peptide within most of the length of the tunnel. However, results indicate that nascent peptides that are protected within the ribosomes vary in length from about 30 to 72 amino acid residues. This suggests that nascent peptides have different conformations. It is hypothesized that folding of the nascent polypeptide into its native conformation starts in the distal portion of the tunnel, and proceeds at the surface of the ribosomal subunit in a depression or bay near the exit opening of the tunnel.

An additional serine residue at the C terminus of rhodanese destabilizes the enzyme.

The rhodanese coding sequence was extended at its 3' end by three base pairs to generate mutants coding for a serine or arginine residue at the carboxyl terminus of the protein. Wild-type and mutant coding sequences were expressed in a cell-free Escherichia coli system by coupled transcription/translation. Predominantly full-length protein was formed in all cases. The amount of protein synthesized was quantified by incorporation of radioactive leucine into polypeptides. Enzymatic activity of in vitro synthesized rhodanese was determined at different temperatures. Specific enzymatic activity was calculated and is assumed to reflect the portion of the protein that is in its native three-dimensional conformation. It was observed that rhodanese extended by one serine at the C terminus lost enzymatic activity when incubated above 30 degrees C, in contrast to wild-type protein or variant rhodanese extended by an arginine residue. Similarly, variant rhodanese with an additional serine residue was more susceptible to urea denaturation than the other two rhodanese species. These results are surprising in light of the crystal structure of the protein.

Expression of different coding sequences in cell-free bacterial and eukaryotic systems indicates translational pausing on Escherichia coli ribosomes.

Five different coding sequences of bacterial or eukaryotic origin in plasmids under the T7 promoter were expressed in a cell-free system derived from Escherichia coli. Translation on E. coli ribosomes resulted in a full-length product only in four of the five coding sequences tested. A unique pattern of less than full-length polypeptides was generated in each case. Many of these polypeptides on E. coli ribosomes reacted with a puromycin derivative, cytidylic acid-puromycin, which was radioactively labeled. Thus these incomplete polypeptides can be defined as nascent peptides bound to the ribosomal P site. Certain nascent peptides could be shifted into full-length protein indicating that they resulted from translational pausing. In contrast to these results, expression of the same coding sequences in a wheat germ or reticulocyte cell-free system resulted in a 80-90% full-length product with no evidence for nascent polypeptides and translational pausing.

Initiation of protein synthesis with fluorophore-Met-tRNA(f) and the involvement of IF-2.

The complicity of initiation factor 2 (IF-2) in causing the observed low incorporation of N-terminal fluorophore from fluorophore-methionyl-tRNA(f) during protein synthesis in an in vitro coupled transcription/translation system was investigated. The low incorporation in comparison to formyl-methionine was not due to the lack of interaction of fluorophore-Met-tRNA(f) with IF-2. Fluorescence measurements of cascade yellow-, eosin-, pyrene-, or coumarin-Met-tRNA(f) determined that all were capable of binding IF-2 at 4 mM Mg(2+) and 37 degrees C. Filter binding assays conducted in the absence of magnesium ions on fMet-tRNA(f), eosin-Met-tRNA(f), and cascade yellow-Met-tRNA(f) confirmed the previously reported value for the dissociation constant of fMet-tRNA(f) of about 1 microM and placed the binding constants for the two fluorophore derivatives about three-fold higher. Binding of the fluorophore-Met-tRNA(f) species to salt-washed ribosomes showed a more significant decrease compared to fMet-tRNA(f). Stimulation in the amount of tRNA bound to the ribosomes upon the addition of IF-2 was observed in each case. All ribosome-bound cascade yellow-Met-tRNA(f) and eosin-Met-tRNA(f) were as puromycin-reactive as fMet-tRNA(f). Cumulatively, the effects observed for the fluorophore-Met-tRNA species in partial reactions of initiation may account for the reduced incorporation of these probes at the N terminus of polypeptides.

Fluorophores at the N terminus of nascent chloramphenicol acetyltransferase peptides affect translation and movement through the ribosome.

Structurally different fluorescent probes were covalently attached to methionyl-tRNA(f) and tested for their incorporation into nascent peptides and full-length protein using an Escherichia coli cell-free coupled transcription/translation system. Bovine rhodanese and bacterial chloramphenicol acetyltransferase (CAT) were synthesized using derivatives of cascade yellow, eosin, pyrene, or coumarin attached to [(35)S]Met-tRNA(f). All of the probes tested were incorporated into polypeptides, although less efficiently when compared with formyl-methionine. Eosin, the largest of the fluorophores used with estimated dimensions of 20 x 11 A, caused the largest reduction in product formed. The rate of initiation was reduced with the fluorophore-Met-tRNA(f) compared with fMet-tRNA(f) with pyrene having the least and eosin the biggest effect. Analysis of the nascent polypeptides showed that the modifications at the N terminus affected the rate at which nascent CAT peptides were elongated causing accumulation of peptides of about 4 kDa, possibly by steric hindrance inside the tunnel within the 50 S ribosomal subunit. Fluorescence measurements indicate that the probe at the N terminus of nascent pyrene-CAT peptides is in a relatively hydrophilic environment. This finding is in agreement with recent data showing cross-linking of the N terminus of nascent peptides to nucleotides of the 23 S ribosomal RNA.

cDNA cloning, characterization, and functional expression of four new monoterpene synthase members of the Tpsd gene family from grand fir (Abies grandis).

Grand fir (Abies grandis) is a useful model system for studying the biochemistry, molecular genetics, and regulation of defensive oleoresin formation in conifers, a process involving both the constitutive accumulation of resin (pitch) in specialized secretory structures and the induced biosynthesis of monoterpenes and sesquiterpenes (turpentine) and diterpene resin acids (rosin) by nonspecialized cells at the site of injury. A similarity-based cloning strategy, employing primers designed to conserved regions of existing monoterpene synthases and anticipated to amplify a 1000-bp fragment, unexpectedly yielded a 300-bp fragment with sequence reminiscent of a terpenoid synthase. Utilization of this amplicon as a hybridization probe afforded four new, full-length cDNA species from a wounded fir stem cDNA library that appeared to encode four distinct monoterpene synthases. Expression in Escherichia coli, followed by enzyme assay with geranyl diphosphate (C(10)), farnesyl diphosphate (C(15)) and geranylgeranyl diphosphate (C(20)), and analysis of the terpene products by chiral phase gas chromatography and mass spectrometry confirmed that these sequences encoded four new monoterpene synthases, including (-)-camphene synthase, (-)-beta-phellandrene synthase, terpinolene synthase, and an enzyme that produces both (-)-limonene and (-)-alpha-pinene. The deduced amino acid sequences indicated these enzymes to be 618 to 637 residues in length (71 to 73 kDa) and to be translated as preproteins bearing an amino-terminal plastid targeting sequence of 50-60 residues. cDNA truncation to delete the transit peptide allowed functional expression of the "pseudomature" forms of these enzymes, which exhibited no change in product outcome as a result of truncation. Sequence comparison revealed that these new monoterpene synthases from grand fir are members of the Tpsd gene subfamily and resemble sesquiterpene (C(15)) synthases and diterpene (C(20)) synthases from conifers more closely than mechanistically related monoterpene synthases from angiosperm species. The availability of a nearly complete set of constitutive and inducible monoterpene synthases from grand fir (now numbering seven) will allow molecular dissection of the resin-based defense response in this conifer species, and detailed study of structure-function relationships among this large and diverse family of catalysts, all of which exploit the same stereochemistry in the coupled isomerization-cyclization reaction.

An attempt to measure the fertility differentials of a semi-urban community of Pondicherry, south India.

PIP: Fertility differentials were measured in a semi-urban community of Pondicherry, South India. Data of the childbearing population, 15-44 years, and the total number of births and deaths and under age 5 children in 1978 and 1982 of the field practice area of the Jawaharlal Institute Urban Health Center (JIUHC) were taken into account. In Pondicherry, the crude birthrate was 32.6/1000 in 1978 and 25.9/1000 in 1982. When the age-specific fertility rates of the 2 years were compared, there was not remarkable difference except for the age group 15-19 years; the age-specific fertility rate was 405.0 in 1978 and 302.0 in 1982, a favorable trend. The total fertility rate was 4.69 in 1978 and 4.28 in 1982. In 1978, the crude rate of natural increase was 15.1; it dropped to 13.6 in 1982. This attempt to measure fertility differentials was useful to assessing population growth and the impact of family welfare services. The data suggest that the population has been inclined to use the family welfare programs.^ieng