Nascent Chain Length Research Articles

Interactions of ribosome nascent chain complexes of the chloroplast-encoded D1 thylakoid membrane protein with cpSRP54.

The mechanisms of targeting, insertion and assembly of the chloroplast-encoded thylakoid membrane proteins are unknown. In this study, we investigated these mechanisms for the chloroplast-encoded polytopic D1 thylakoid membrane protein, using a homologous translation system isolated from tobacco chloroplasts. Truncated forms of the psbA gene were translated and stable ribosome nascent chain complexes were purified. To probe the interactions with the soluble components of the targeting machinery, we used UV-activatable cross-linkers incorporated at specific positions in the nascent chains, as well as conventional sulfhydryl cross-linkers. With both cross-linking approaches, the D1 ribosome nascent chain was photocross-linked to cpSRP54. cpSRP54 was shown to interact only when the D1 nascent chain was still attached to the ribosome. The interaction was strongly dependent on the length of the nascent chain that emerged from the ribosome, as well as the cross-link position. No interactions with soluble SecA or cpSRP43 were found. These results imply a role for cpSRP54 in D1 biogenesis.

Secretory proteins move through the endoplasmic reticulum membrane via an aqueous, gated pore

The environment of secretory proteins undergoing translocation across the ER membrane was determined by incorporating fluorescent probes into nascent chains during translation. Dyes were positioned at various locations across the entire bilayer and inside the ribosome, and in each case the probes were in an aqueous milieu, as shown both by their fluorescence lifetimes and by collisional quenching of their fluorescence by iodide ions introduced into the ER lumen. The nascent chain therefore occupies an aqueous pore that spans the entire membrane. Since the pore is sealed off from the cytoplasm, cotranslational translocation is effected topographically. This pore is not open to the lumen after targeting is completed; it opens only after the nascent chain length reaches about 70 residues.

Single-stranded DNA-dependent ATPase from HeLa cells that stimulates DNA polymerase .alpha.-primase activity: purification and characterization of the ATPase

A single-stranded DNA-dependent ATPase that cofractionates during the early stages of purification of a multiprotein DNA polymerase alpha complex from HeLa cells has been purified to homogeneity. The ATPase is part of a 16S multienzyme DNA polymerase alpha complex that is fully active in SV40 DNA replication in vitro. The ATPase hydrolyzes ATP to ADP in a reaction that is completely dependent on the presence of DNA. DNA in single-stranded form is strongly preferred as a cofactor, and polydeoxynucleotides with adenine or thymidine residues are highly effective. Glycerol gradient sedimentation showed that the purified ATPase sedimented at an s20,w of 7 S, and polyacrylamide gel electrophoresis under denaturing conditions reveals two polypeptides with relative molecular weights of 83,000 and 68,000. Both of these polypeptides have purine nucleotide binding sites as revealed by photoaffinity cross-linking experiments. ATP binds to the two subunits more efficiently than GTP, and CTP or UTP does not cross-link with the two polypeptides. DNA synthesis catalyzed by purified HeLa cell DNA polymerase alpha-primase is stimulated in the presence of ATPase and ATP at an optimum concentration of 2 mM. Analysis of the DNA product by gel electrophoresis indicates that with poly(dT) but not phage M13 DNA as template the ATPase overcomes a lag and decreases the length of nascent DNA chains synthesized by the DNA polymerase alpha-primase complex.

The affinity of signal recognition particle for presecretory proteins is dependent on nascent chain length.

We have developed an assay in which incomplete preprolactin chains of varying lengths are targeted to the endoplasmic reticulum (ER) membrane in an elongation independent manner. The reaction had the same molecular requirements as nascent chain translocation across the ER membrane, namely, it was signal recognition particle (SRP) dependent, and required the nascent chain to be present as peptidyl tRNA (i.e. most likely ribosome associated) and to have its signal sequence exposed outside the ribosome. We found that the efficiency of the targeting reaction dropped dramatically as the chains grew longer than 140 amino acids in length, which probably reflected a decrease in affinity of the nascent chain-ribosome complex for SRP. Thus at physiological SRP concentrations (10 nM) there appears a sharp cut-off point in the ability of these chains to be targeted, while at high SRP concentrations (270 nM) all chains could be targeted. In kinetic experiments, high concentrations of SRP were found to change the time in elongation after which translocation of the nascent polypeptide could no longer occur.

The role of palindromic and non-palindromic sequences in arresting DNA synthesis in vitro and in Vivo

The nature of specific DNA sequences that arrest synthesis by mammalian DNA polymerase α in vitro was analyzed using circular, single-stranded M13 or φX174 virion DNA templates annealed to a unique, terminally labeled, DNA primer. This method rigorously defined both the starting nucleotide position and the direction of synthesis, as well as making the amount of radioactivity proportional to the number rather than the length of nascent DNA chains. The precise nucleotide locations of arrest sites were determined over templates with complementary sequences by cloning unique DNA restriction fragments into M13 DNA and isolating virions containing either the Watson or Crick strand. Results were correlated with the locations of palindromic (self-complementary) sequences, repeated sequences, and repeated sequences with mirror-image orientation. Two classes of DNA synthesis arrest sites were identified, distinct in structure but equivalent in activity. Class I sites consisted of palindromic sequences that formed a stable hairpin structure in solution and arrested DNA polymerase on both complementary templates. The polymerase stopped precisely at the base of the duplex DNA stem, regardless of the direction from which the enzyme approached. Class II sites consisted of non-palindromic sequences that could not be explained by either secondary structure or sequence symmetry elements, and whose complementary sequence was not an arrest site. Size limits, orientation and some sequence specificity for arrest sites were suggested by the data. Arrest sites were also observed in vivo by mapping the locations of 3′-end-labeled nascent simian virus 40 DNA strands throughout the genome. Arrest sites closest to the region where termination of replication occurs were most pronounced, and the locations of 80% of the most prominent sites appeared to be recognized by α-polymerase on the same template in vitro. However, class I sites were not identified in vivo, suggesting that palindromic sequences do not form hairpin structures at replication forks.

Specific sequences in native DNA that arrest synthesis by DNA polymerase alpha.

The effect of the DNA sequence of a template on the progress of DNA polymerase alpha was determined at the resolution of single nucleotides by using single-stranded, circular phi X174 DNA as a template and unique phi X 174 ONA fragments terminally labeled at their 3'-ends as primers. Therefore, the amount of radioactivity observed was always proportional to the number and not the length of nascent DNA chains. Extension of a primer by alpha-polymerase revealed that 3'-ends of nascent DNA chains accumulated at specific arrest sites consisting of GC-rich sequences of 1-8 bases distributed nonuniformly along the template with intervening sequences of 0-140 bases. The precise location and composition of these sites were determined by DNA sequencing methods, but a consensus sequence was not detected. However, the same pattern of arrest sites recognized by DNA polymerase alpha from CV-1 cells also arrested alpha-polymerase from HeLa cells, calf thymus tissue, and phage T4 DNA polymerase. An exception was DNA polymerase I from Escherichia coli which was insensitive to arrest signals. Analysis of recombinant DNA templates containing phi X174 DNA inserted into M13 DNA revealed that arrest sites were defined exclusively by those sequences within 24 bases upstream and 140 bases downstream of the arrest point; long range interactions between template sequences were not involved. One of the strongest arrest sites was located in the first two nucleotides at the base of the stem on the primer-proximal side of a stable hairpin structure, suggesting that such structures arrest the polymerase. However, only 28% of all arrest site nucleotides were found in this position; the rest were up to 25 bases upstream from any computer-predicted hairpin. Therefore, all arrest sites cannot be defined by secondary structure alone, although proximity to secondary structures may amplify normal variations in the rate of DNA elongation caused by primary sequences.

The 5′→3′ polarity of the xenopus ribosomal RNA precursor molecule

Evidence is presented that in Xenopus laevis the 18S rRNA sequence is proximal to the 5′ end of the rRNA precursor molecule and the 28S sequence is proximal to the 3′ end. This assignment was made by digesting amplified ribosomal gene transcription complexes with EcoRI restriction endonuclease and spreading the cleaved transcription complexes for electron microscopy. From the known location of the EcoRI cutting sites within the transcribed region and the length of nascent chains on the cleaved transcription complexes, it was possible to make an unambiguous assignment of polarity.

Characterisation of RNA synthesis in vitro neuron-rich, oligodendroglial and liver nuclei.

In neurone‐rich nuclei isolated from the adult guinea‐pig cerebral cortex, DNA‐dependent RNA polymerase B reaction is several times more active than in liver nuclei. This may be attributable to the unusual wealth of euchromatin in mature neuronal nuclei. Nuclear RNA labelled in vitro was hydrolysed by alkali, and the number as well as the average length of nascent RNA chains were determined by measuring [3H]uridine and [3H]UMP/[3H]uridine ratio, respectively. In neurone‐rich nuclei, the number of growing RNA chains is greater, and the average chain length longer than in liver nuclie. In oligodendroglial nuclei which exhibit RNA polymerase B reaction of intermediate activity, the number of growing RNA chains is found comparable to, but the average chain length longer, than that in liver nuclei. Thus, the presence of a greater number of growing RNA chains appears to be a unique feature of neuronal nuclei. In neuronal chromatin, mean residue ellipticity in circular dichroism spectrum in the wavelength range of 260–300 nm is significantly higher than in liver and oligodendroglial chromatins, and this is in apparent correlation with the number, rather than the average length, of growing RNA chains. Digestion of chromatin with trypsin leads to reversion of the circular dichroism spectrum toward that of free DNA, and completely abolishes the difference in ellipticity pattern that is observed among native neurone‐rich, oligodendroglial and liver chromatins. In liver nuclei, RNA polymerase B reaction is greatly enhanced with trypsin, which is primarily attributable to the facilitated elongation of RNA chains that is otherwise restricted. In neuronal nuclei where the steric restriction imposed on RNA synthesis is though to be intrinsically weakened, digestion with trypsin is more than necessary in disclosing the RNA polymerase B activity. RNA synthesis in neuronal and oligodendroglial nuclei in vitro is discussed in correlation with that reported in vivo.

Delayed removal of N-terminal methionine from nascent globin chains in sickle-cell anemia reticulocytes.

Synthetic alphaT1 and betaT1, the N-terminal tryptic peptides of alpha-chain and beta-chain of hemoglobin, and MetalphaT1 and MetbetaT1, peptides in which N-terminal methionyl residues are peptide-bonded to alphaT1 and betaT1, were prepared by the solid-state method of Merrifield. These synthetic peptides were used to establish conditions for chromatographic purification and analysis. When tryptic digests of nascent globin chains from rabbit and sickle-cell anemia reticulocytes incubated with (35)S- and (3)H-labeled amino acids were analyzed, radioactivity not present in tryptic digests of labeled hemoglobin appeared at the elution positions of MetalphaT1 and MetbetaT1. The fraction of nascent chains with N-terminal methionine was higher in sickle-cell anemia reticulocytes than in rabbit reticulocytes. If rate of peptide-chain elongation in polysomes is uniform, nascent human chains must attain a greater length before removal of the initial methionyl residue. Length of nascent chain at time of removal was calculated from two independent sets of data, one obtained from [(35)S]methionine incorporation into MetalphaT1, MetbetaT1, alphaT5, and betaT5, and the other obtained from [(3)H]lysine and [(3)H]valine incorporation into MetbetaT1 and betaT1.