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Biomedical subjects

M Kozak

Publications and source records attributed to M Kozak.

At least 91 records · Page 5Linked to original sources
PubMed1981

Twelve months' experience with continuous ambulatory and intermittent peritoneal dialysis.

After a one-year experience with a continuous ambulatory and long-term intermittent peritoneal dialysis (CAPD and IPD, respectively) program in a Veterans Administration hospital, both forms of dialysis provided excellent biochemical control of the patients' conditions. The major drawback to peritoneal dialysis as opposed to hemodialysis is the high rate of rehospitalization resulting from peritonitis or problems related to the peritoneal catheter. The incidence of peritonitis was one episode per 4.1 patient months in CAPD and one episode per 7.3 patient months in IPD. Recurrent episodes of peritonitis in a given patient were associated with a decrease in the serum albumin level. Blood values for BUN, creatinine, serum electrolytes, calcium, and phosphorus, however, were not altered. To date, CAPD appears to be an effective alternative form of dialytic therapy.

Adult↗
PubMed1980

Role of ATP in binding and migration of 40S ribosomal subunits.

Two assays have been devised to demonstrate ATP-dependent migration of 40S ribosomal subunits on messenger RNA. The first is a two-step runoff assay. Reovirus mRNA was initially loaded with 40S subunits by incubation with wheat germ ribosomes in the presence of the antibiotic edeine. During the second phase of the incubation, in which further attachment of ribosomes was inhibited, the preformed complexes were shown to dissociate (presumably by runoff) only if ATP was included in the reaction. A more direct demonstration of ATP-dependent migration of 40S subunits was carried out using 3' end-labeled brome mosaic virus mRNA. In the presence of edeine and ATP, 40S ribosomal subunits were shown to advance all the way to the 3' end of the message, as shown by protection of the labeled 3'-proximal segment against nuclease digestion. Depletion of ATP by the addition of hexokinase prevented this migration. A variety of observations has raised the possibility that attachment of eucaryotic ribosomes to messenger RNA proceeds via a "scanning mechanism." The hypothesis is that a 40S subunit binds initially at or near the 5' terminus of the message and subsequently migrates toward the interior, stopping when it encounters the first AUG triplet. If migration of 40S subunits requires ATP, as the present studies suggest, the scanning mechanism predicts that in a system depleted of ATP a single 40S ribosome should be trapped near the 5' terminus of the message--upstream of the AUG initiator codon. This prediction was confirmed by analyzing binding of wheat germ ribosomes to a synthetic ribopolymer in which the 5'-proximal region (lacking AUG codons) and the AUG-containing segment near the 3' end of the molecule were differentially labeled.

Adenosine Triphosphate↗
PubMed1980

Influence of mRNA secondary structure on binding and migration of 40S ribosomal subunits.

Reovirus messenger RNA was modified by reaction with bisulfite (in denaturing conditions) or by incorporation of IMP in place of GMP, thereby irreversibly unfolding the mRNA. Messenger RNA in which the secondary structure was weakened or abolished retained the ability to bind to wheat germ ribosomes, suggesting that conformational features around the AUG codon are not required for ribosome recognition of mRNA. Ribosomes were not able to attach (directly) to spurious internal sites, even in extensively unfolded RNA, indicating that the monocistronic character of eucaryotic messages (in which initiation is limited to a single 5' proximal site) is not simply due to conformational masking of all the internal AUG codons. The secondary structure in eucaryotic messages does contribute to the fidelity of the translation process, however, because when 40S ribosomal subunits were incubated with denatured mRNA they failed to stop at the 5' proximal AUG codon. Extensive migration beyond the 5' region occurred when 40S ribosomes (in the absence of 60S subunits) attached to unfolded mRNA, implying that the secondary structure in native mRNA facilitates correct translation by impeding migration of 40S subunits beyond the 5' proximal initiation region. Secondary structure in mRNA may also modulate the efficiency of translation. Studies with BrUMP-substituted mRNA, in which the secondary structure is enhanced, suggested that the efficiency of mRNA binding to ribosomes decreases as the stability of the secondary structure increases.

Codon↗
PubMed1980

Binding of wheat germ ribosomes to fragmented viral mRNA.

The specificity of binding of wheat germ ribosomes to mRNA was greatly altered by cleavage of the message. Fragmentation of reovirus mRNA allowed wheat germ ribosomes to bind and protect a variety of internal sequences which were not accessible to ribosomes in the intact message. In experiments using the polycistronic mRNA from bacteriophage R17, wheat germ ribosomes bound preferentially at the beginning of the lysis peptide and synthetase cistrons, and at a third site which may be derived from the C-terminal region of the A protein cistron. This result is similar to that reported previously in a mammalian translational system (J.F. Atkins et al., Cell 18:246-256, 1979) except that, in the present study, limited cleavage of the phage RNA was necessary to activate these sites. More extensive fragmentation of R17 RNA permitted wheat germ ribosomes to bind and protect a great many additional sites. Thus, presence of an (exposed) 5'-terminus on an RNA molecule appears to be necessary and sufficient for attachment of eucaryotic ribosomes.

Bacteriophages↗
PubMed1979

Migration of 40 S ribosomal subunits on messenger RNA when initiation is perturbed by lowering magnesium or adding drugs.

Migration of 40 S ribosomal subunits on messenger RNA, detected previously in experiments using the antibiotic edeine (Kozak, M., and Shatkin, A.J. (1978) J. Biol. Chem. 253, 6568-6577) has now been observed in the presence of other inhibitors of initiation. 40 S subunit migration has been detected in both wheat germ and reticulocyte lysates treated with edeine, pactamycin, or sodium fluoride. The variety of structurally unrelated inhibitors that mediate this effect argues against the interpretation that migration is a drug-induced artifact. Indeed, limited migration of 40 S ribosomes occurs upon simply lowering the magnesium concentration, in the absence of inhibitors. Thus, migration seems to be an inherent property of 40 S ribosomal subunits and might be involved in the mechanism by which eukaryotic ribosomes select initiation sites in messenger RNA.

Animals↗
PubMed1978

Migration of 40 S ribosomal subunits on messenger RNA in the presence of edeine.

The antibiotic edenine induces binding of multiple 40 S ribosomes to reovirus messenger RNAs, producing complexes that sediment rapidly in glycerol gradients. Rapidly sedimenting complexes were also obtained with tobacco mosaic virus RNA and rabbit globin mRNA in the presence of edeine. Following ribonuclease digestion of the heavy complexes, nuclease-resistant 32P-labeled reovirus fragments protected by 40 S ribosomes in the presence of edeine were recovered and fingerprinted. The sequence complexity of the protected material supports the interpretation that 40 S subunits are distributed at many internal sites in each messenger RNA. Additional experiments indicate that binding of the multiple 40 S subunits occurs from a single "entry site" which involves the 5' terminus of the message. This, in turn, implies that in the presence of edeine 40 S ribosomes are able to move along the mRNA chain, attaching initially near the 5' end, then advancing to make room for the next subunit. We suggest that in the absence of antibiotics, also, a 40 S ribosome might bind near the 5' terminus and then advance, stopping where it encounters the first AUG triplet. The effect of edeine might be to interfere with the AUG recognition process, thus allowing the 40 S ribosome to continue unhalted along the message. The present experiments with edeine provide the first direct evidence that 40 S ribosomal subunits are capable of moving along the mRNA chain.

Anti-Bacterial Agents↗
PubMed1976

Characterization of ribosome-protected fragments from reovirus messenger RNA.

The 5'-terminal methylated cap (m7G(5')ppp(5')Gm) in reovirus messenger RNA comprises part of the ribosomes binding site, since attachment of 40 S wheat germ ribosomal subunits to reovirus small (s), medium (m), and large (l) RNA classes conferred almost complete protection of the cap against RNase digestion. After joining of the 60 S ribosomal subunits, however, the cap continued to be protected against T1 RNase within the 80 S initiation complexes formed with only some messenger species; namely the three l-messages, one of the m-messages, and one or two of the s-messages. When protected fragments were recovered from 40 S and 80 S complexes and tested for ability to rebind to ribosomes those fragments which retained the cap were able to rebind most efficiently. The protected fragments recovered from 40 S initiation complexes with several of the s- and m-RNA species were larger than the messenger fragments recovered from 80 S complexes. The medium size class of reovirus RNA, which consists of three messenger species, gave rise to three discrete 5'-terminal fragments after digestion of 40 S complexes with T1 RNase, and to three somewhat smaller fragments after T1 RNase digestion of 80 S complexes. Fingerprints of the T1 oligonucleotides derived from these fragments are consistent with the interpretation that each messenger species within the m-RNA class gives rise to a protected fragment of a unique size and that, with each message, there is extensive overlap between the regions of the message protected by 40 S and 80 S ribosomes. The ratio of the three protected fragments recovered from 40 S complexes with m-RNA was highly reproducible under a given set of binding conditions, but could be shifted by varying the messenger/ribosome ratio in the binding reaction. Thus, one of the fragments, which was preferentially recovered when the ribosome concentration was limiting, could be tentatively identified as the binding site of the most efficiently translated message within the m-RNA class.

Oligoribonucleotides↗