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M Kozak

Publications and source records attributed to M Kozak.

At least 55 records · Page 3Linked to original sources
PubMed1995

Adherence to the first-AUG rule when a second AUG codon follows closely upon the first.

The rule that eukaryotic ribosomes initiate translation exclusively at the 5' proximal AUG codon is abrogated under rare conditions. One circumstance that has been suggested to allow dual initiation is close apposition of a second AUG codon. A possible mechanism might be that the scanning 40S ribosomal subunit flutters back and forth instead of stopping cleanly at the first AUG. This hypothesis seems to be ruled out by evidence presented herein that in certain mRNAs, the first of two close AUG codons is recognized uniquely. To achieve this, the 5' proximal AUG has to be provided with the full consensus sequence; even small departures allow a second nearby AUG codon to be reached by leaky scanning. This context-dependent leaky scanning unexpectedly fails when the second AUG codon is moved some distance from the first. A likely explanation, based on analyzing the accessibility of a far-downstream AUG codon under conditions of initiation versus elongation, is that 80S elongating ribosomes advancing from the 5' proximal start site can mask potential downstream start sites.

Adenine↗
PubMed1995

Oxidatively modified LDL contains phospholipids with platelet-activating factor-like activity and stimulates the growth of smooth muscle cells.

Oxidative modification of lipoproteins is believed to be important in the genesis of atherosclerosis. We established cultures of smooth muscle cells (SMC) and exposed them to native LDL or oxidized LDL. Oxidized LDL, but not native LDL, was mitogenic as measured by incorporation of [3H]-thymidine into DNA. This effect was concentration dependent, averaged 288% of control, and was blocked by a platelet-activating factor (PAF) receptor antagonist. We hypothesized that phospholipids with PAF-like activity were generated during the oxidation of LDL. To test this hypothesis we extracted phospholipids from copper-oxidized LDL and assayed for PAF-like activity. Phospholipids extracted from oxidized LDL and purified by HPLC induced neutrophil adhesion equivalent to PAF (10 nM) and were mitogenic for smooth muscle cells. These effects were not seen with phospholipids extracted from native LDL and were blocked by two structurally different, competitive antagonists of the PAF receptor. The effects of these lipids were also abolished by pretreating them with PAF acetylhydrolase. Finally, we used Chinese hamster ovary cells that had seen stably transfected with a cDNA for the PAF receptor to confirm that phospholipids from oxidized LDL act via this receptor. We found that PAF (control) and the oxidized phospholipids each induced release of arachidonic acid from the transfected cells, but had no effect on wildtype Chinese hamster ovary cells, which lack the PAF receptor. This effect was also blocked by a PAF receptor antagonist. Thus, phospholipids generated during oxidative modification of LDL may participate in atherosclerosis by stimulating SMC proliferation and leukocyte activation.

Animals↗
PubMed1995

The Churg-Strauss syndrome. A case report with angiographically documented coronary involvement and a review of the literature.

The Churg-Strauss syndrome (CSS) is an unusual disease that presents as a systemic vasculitis and peripheral eosinophilia in a patient with chronic atopic disease. Although often not prominent on initial presentation, cardiac involvement is a major cause of morbidity and mortality in patients with CSS. We report a case of a young woman with CSS who had a myocardial infarction. Coronary arteriography was performed for recurrent chest pain and demonstrated diffuse vasculopathy consistent with vasculitis in CSS. We have also included a review of the literature on cardiac involvement in CSS.

Adult↗
PubMed1994

Features in the 5' non-coding sequences of rabbit alpha and beta-globin mRNAs that affect translational efficiency.

The 5' non-coding sequence of rabbit beta-globin mRNA was mutagenized in an attempt to identify structural features that might contribute to the ability to support translation in an homologous rabbit reticulocyte lysate. Translational efficiency was not reduced by substitutions introduced in nearly every position of the beta-globin leader sequence, suggesting that the 5' non-coding domain of this highly efficient mRNA contains no special effector motifs. Instead, efficient translation appears to require only a moderately long leader sequence devoid of secondary structure, especially near the 5' end. Consistent with that interpretation, substitutions in several positions actually improved translation relative to the wild-type beta-globin leader sequence; experimental assessment of the secondary structure of these derivatives revealed a perfect inverse correlation between secondary structure content and translational efficiency. Other experiments probed the structural basis for the long-noted difference in translational efficiency between rabbit alpha and beta-globin mRNAs, a difference that was reproduced here using only the 5' non-coding domains of those mRNAs. The possibility that translation of ribosomal protein mRNAs might be modulated by a mechanism similar to that of alpha-globin mRNA is discussed. Because the beta-globin leader sequence has been incorporated into some popular expression vectors, and because globin genes are targets for gene therapy, this analysis of how globin mRNA leader sequences function in translation and how they can be improved may have practical applications.

Animals↗
PubMed1992

A consideration of alternative models for the initiation of translation in eukaryotes.

Although recent biochemical and genetic investigations have produced some insights into the mechanism of initiation of translation in eukaryotic cells, two aspects of the initiation process remain controversial. One unsettled issue concerns a variety of functions that have been proposed for mRNA binding proteins, including some initiation factors. The need to distinguish between specific and nonspecific binding of proteins to mRNA is discussed herein. The possibility that certain initiation factors might act as RNA helicases is evaluated along with other ideas about the functions of mRNA- and ATP-binding factors. A second controversial issue concerns the universality of the scanning mechanism for initiation of translation. According to the conventional scanning model, the initial contact between eukaryotic ribosomes and mRNA occurs exclusively at the 5' terminus of the message, which is usually capped. The existence of uncapped mRNAs among a few plant and animal viruses has prompted a vigorous search for other modes of initiation. An "internal initiation" mechanism, first proposed for picornaviruses, has received considerable attention. Although a large body of evidence has been adduced in support of such a mechanism, many of the experiments appear flawed or inconclusive. Some suggestions are given for improving experiments designed to test the internal initiation hypothesis.

Eukaryotic Cells↗
PubMed1991

An analysis of vertebrate mRNA sequences: intimations of translational control.

Five structural features in mRNAs have been found to contribute to the fidelity and efficiency of initiation by eukaryotic ribosomes. Scrutiny of vertebrate cDNA sequences in light of these criteria reveals a set of transcripts--encoding oncoproteins, growth factors, transcription factors, and other regulatory proteins--that seem designed to be translated poorly. Thus, throttling at the level of translation may be a critical component of gene regulation in vertebrates. An alternative interpretation is that some (perhaps many) cDNAs with encumbered 5' noncoding sequences represent mRNA precursors, which would imply extensive regulation at a posttranscriptional step that precedes translation.

Animals↗
PubMed1991

A short leader sequence impairs the fidelity of initiation by eukaryotic ribosomes.

The functional consequences of unusually short 5' noncoding sequences on eukaryotic mRNAs are explored here by using an in vitro transcription and translation system. As the distance of the first AUG codon from the m7G cap was decreased from 32 to 3 nucleotides, the yield of protein initiated from the first AUG codon progressively decreased, with a corresponding increase in initiation from the second AUG codon. The leakiness attributable to a too-short leader sequence was offset, however, by introducing secondary structure downstream from the first AUG codon.

Base Sequence↗
PubMed1991

Effects of long 5' leader sequences on initiation by eukaryotic ribosomes in vitro.

Lengthening the 5' noncoding sequence on SP6-derived transcripts can increase their translational efficiency by an order of magnitude under some conditions of translation in reticulocyte lysates. This effect was observed upon reiterating three different synthetic oligonucleotides, the sequences of which were designed simply to preclude secondary structure. It seems unlikely that such arbitrarily designed sequences are recognized by sequence-specific translational enhancer proteins. Rather, long 5' leader sequences appear to accumulate extra 40S ribosomal subunits, which may account for their translational advantage. The buildup of 40S subunits on long, unstructured leader sequences is predicted by the scanning model for initiation. Leader sequences such as these may be ideal for in vitro expression vectors.

Base Sequence↗
PubMed1990

Downstream secondary structure facilitates recognition of initiator codons by eukaryotic ribosomes.

Recognition of an AUG initiator codon in a suboptimal context improves when a modest amount of secondary structure is introduced near the beginning of the protein-coding sequence. This facilitating effect depends on the position of the downstream stem-loop (hairpin) structure. The strongest facilitation is seen when the hairpin is separated from the preceding AUG codon by 14 nucleotides. Because 14 nucleotides corresponds to the approximate distance between the leading edge of the ribosome and its AUG-recognition center as measured by ribonuclease protection experiments, a likely explanation for the enhancing effect of a downstream hairpin is that secondary structure slows scanning, thereby providing more time for recognition of the AUG codon, and the facilitation is greatest when the 40S ribosome stalls with its AUG-recognition center directly over the AUG. The variable ability of mammalian ribosomes to initiate at non-AUG codons in vitro is also explicable by the presence or absence of a stem-loop structure just downstream from the alternative initiator codon. This may be relevant to recent reports of adventitious upstream initiation events at non-AUG codons in some vertebrate mRNAs that have structure-prone, G + C-rich leader sequences.

Animals↗
PubMed1989

The scanning model for translation: an update.

The small (40S) subunit of eukaryotic ribosomes is believed to bind initially at the capped 5'-end of messenger RNA and then migrate, stopping at the first AUG codon in a favorable context for initiating translation. The first-AUG rule is not absolute, but there are rules for breaking the rule. Some anomalous observations that seemed to contradict the scanning mechanism now appear to be artifacts. A few genuine anomalies remain unexplained.

Animals↗
PubMed1989

Context effects and inefficient initiation at non-AUG codons in eucaryotic cell-free translation systems.

The context requirements for recognition of an initiator codon were evaluated in vitro by monitoring the relative use of two AUG codons that were strategically positioned to produce long (pre-chloramphenicol acetyl transferase [CAT]) and short versions of CAT protein. The yield of pre-CAT initiated from the 5'-proximal AUG codon increased, and synthesis of CAT from the second AUG codon decreased, as sequences flanking the first AUG codon increasingly resembled the eucaryotic consensus sequence. Thus, under prescribed conditions, the fidelity of initiation in extracts from animal as well as plant cells closely mimics what has been observed in vivo. Unexpectedly, recognition of an AUG codon in a suboptimal context was higher when the adjacent downstream sequence was capable of assuming a hairpin structure than when the downstream region was unstructured. This finding adds a new, positive dimension to regulation by mRNA secondary structure, which has been recognized previously as a negative regulator of initiation. Translation of pre-CAT from an AUG codon in a weak context was not preferentially inhibited under conditions of mRNA competition. That result is consistent with the scanning model, which predicts that recognition of the AUG codon is a late event that occurs after the competition-sensitive binding of a 40S ribosome-factor complex to the 5' end of mRNA. Initiation at non-AUG codons was evaluated in vitro and in vivo by introducing appropriate mutations in the CAT and preproinsulin genes. GUG was the most efficient of the six alternative initiator codons tested, but GUG in the optimal context for initiation functioned only 3 to 5% as efficiently as AUG. Initiation at non-AUG codons was artifactually enhanced in vitro at supraoptimal concentrations of magnesium.

Animals↗
PubMed1989

Circumstances and mechanisms of inhibition of translation by secondary structure in eucaryotic mRNAs.

This paper describes in vitro experiments with two types of intramolecular duplex structures that inhibit translation in cis by preventing the formation of an initiation complex or by causing the complex to be abortive. One stem-loop structure (delta G = -30 kcal/mol) prevented mRNA from engaging 40S subunits when the hairpin occurred 12 nucleotides (nt) from the cap but had no deleterious effect when it was repositioned 52 nt from the cap. This result confirms prior in vivo evidence that the 40S subunit-factor complex, once bound to mRNA, has considerable ability to penetrate secondary structure. Consequently, translation is most sensitive to secondary structure at the entry site for ribosomes, i.e., the 5' end of the mRNA. The second stem-loop structure (hp7; delta G = -61 kcal/mol, located 72 nt from the cap) was too stable to be unwound by 40S ribosomes, hp7 did not prevent a 40S ribosomal subunit from binding but caused the 40S subunit to stall on the 5' side of the hairpin, exactly as the scanning model predicts. Control experiments revealed that 80S elongating ribosomes could disrupt duplex structures, such as hp7, that were too stable to be penetrated by the scanning 40S ribosome-factor complex. A third type of base-paired structure shown to inhibit translation in vivo involves a long-range interaction between the 5' and 3' noncoding sequences.

Animals↗
PubMed1988

Neonatal testosterone administration, but not in utero contiguity to males, augments the display of male sexual behavior by testosterone-treated adult female mice.

Male copulatory behavior of adult female mice given slow-release capsules of testosterone was examined in animals that developed in utero contiguous to two males (mFm) or to two females (fFf). Other females of unspecified uterine position which were injected with testosterone propionate on the day of birth as well as intact males also were examined. mFm and fFf females did not differ on any measure; latency to the first mount, number of mount bouts, number of mount bouts with genital thrusting. The perinatally androgenized females exhibited more mount bouts and more bouts accompanied by genital thrusting than did mFm and fFf subjects. The former also displayed more mount bouts with thrusting on the second pair of tests than males. Lastly, a greater proportion of perinatally androgenized females than mFm or fFf animals displayed male sexual behavior two weeks following removal of the testosterone-containing capsule.

Animals↗