The mechanism of ribosome binding to eucaryotic mRNAs is not well understood, but it requires the participation of eucaryotic initiation factors eIF-4A, eIF-4B, and eIF-4F and the hydrolysis of ATP. Evidence has accumulated in support of a model in which these initiation factors function to unwind the 5'-proximal secondary structure in mRNA to facilitate ribosome binding. To obtain direct evidence for initiation factor-mediated RNA unwinding, we developed a simple assay to determine RNA helicase activity, and we show that eIF-4A or eIF-4F, in combination with eIF-4B, exhibits helicase activity. A striking and unprecedented feature of this activity is that it functions in a bidirectional manner. Thus, unwinding can occur either in the 5'-to-3' or 3'-to-5' direction. Unwinding in the 5'-to-3' direction by eIF-4F (the cap-binding protein complex), in conjunction with eIF-4B, was stimulated by the presence of the RNA 5' cap structure, whereas unwinding in the 3'-to-5' direction was completely cap independent. These results are discussed with respect to cap-dependent versus cap-independent mechanisms of ribosome binding to eucaryotic mRNAs.A critical step in eucaryotic protein biosynthesis is binding of the small (40S) ribosomal subunit to mRNA (36,39). This step is rate limiting in translation initiation (25) and is a key target for regulation (reviewed in reference 53), but the mechanism of this process is poorly understood. Two pathways for the binding of 40S ribosomal subunits to mRNA have been described, which differ in their requirement for the cap structure. The 5' cap structure, m7GpppX (where X is any nucleotide) is a nearly ubiquitous feature of all eucaryotic mRNAs (49). Evidence indicates that translation initiation of the majority of eucaryotic mRNAs is accomplished in a cap-enhanced manner, whereby 40S ribosomal binding to mRNA is facilitated by the cap structure (reviewed in references 3 and 50). Recently, it has been shown that poliovirus (42) and encephalomyocarditis virus (26) mRNAs, which are naturally uncapped (13,21,38), initiate translation by a different mechanism. In this case, the 40S subunit binds directly to an internal element on the picornavirus 5' untranslated region, by-passing upstream sequences and the requirement for the cap structure.Although the two initiation pathways are mechanistically distinguishable, they nevertheless require a similar set of initiation factors (eIFs [57]) to bind 40S ribosomal subunits to mRNA. Cap-stimulated mRNA binding to the small ribosomal subunit requires at least three initiation factors, eIF-4A, eIF-4B, and eIF-4F, in addition to the hydrolysis of ATP (reviewed in references 10, 44, and 53). eIF-4F is a multisubunit complex consisting of three major polypeptides of 24, 50, and 220 kilodaltons (kDa) (8,20,59). The 24-kDa polypeptide is the cap-binding subunit, which also exists in a free form, termed eIF-4E (55). The 50-kDa polypeptide is a structural variant of free eIF-4A (8,20). Although eIF-4F contains an eIF-4A subunit which is almost identical to...

authors request that the sequence shown in Fig. 2 of the original report should be corrected as follows. As shown schematically here in Fig. 1 A, nucleotides 1452-1504 of the original report have to be replaced by a novel sequence shown here in Fig. 1B. This correction pertains to the 5Ј-flanking region of the gene and does not affect the coding region nor any of the conclusions drawn in the original report. The fact that there was a missing sequence element was discovered and pointed out to us by Tracy L. Bale in the laboratory of Daniel M. Dorsa, Departments of Psychiatry and Behavioral Sciences and Pharmacology, University of Washington, Seattle. As illustrated in Fig. 1 A, the novel sequence has to be inserted at the location of a dinucleotide repeat, (GT) 26 , located 89 nucleotides 5Ј to the main transcriptional initiation site. Resequencing of a newly generated phage subclone as well as Southern blot and PCR analyses (not shown) confirmed that the sequence presented here is indeed part of the genomic sequence. Since this novel sequence element is itself flanked by two dinucleotide repeats, (GT) 20 and (GT) 24 , respectively, a likely explanation is that this segment was spliced out during subcloning due to recombination between the two dinucleotide repeats. This idea is further supported by the fact that dinucleotide repeats that have the potential of forming Z-DNA structures have been shown to enhance recombination in extrachromosomal DNA up to 20-fold (1). Despite the recurrence of dinucleotide repeats around chromosomal rearrangement breakpoints, their role in mediating recombination on intact chromosomes remains, however, uncertain (2).We thank Tracy L. Bale and Daniel M. Dorsa for pointing out the error and for their help and collaboration in its correction. Fig. 3, A and C, are of (M ϩ 33H) 33ϩ .17. Mariman, E. C. M., Broers, C. A. M., Claesen, C. A.

It is well established that uterine oxytocin receptors (OTRs) are strongly up-regulated immediately before parturition as well as in response to estrogen (E2) administration. Progesterone (P4), on the other hand, induces a rapid down-regulation. We recently cloned the rat OTR gene and characterized its expression in the rat uterus. In this study, we examined the regulation of OTR messenger RNA (mRNA) levels in rat uterus during pregnancy, the estrous cycle, and in response to gonadal steroid treatment. OTR mRNA levels increased more than 25-fold during gestation: 4.5-fold during the first 21 days and 6-fold within 24 h between day 21 and the onset of parturition. Uterine OTR mRNA levels fell rapidly by 85% within 24 h following parturition. By in situ hybridization, OTR mRNA was localized specifically to the longitudinal and circular layers of the myometrium but was not detected in the endometrium. During the estrous cycle, OTR mRNA levels increased 2-fold between metestrus and proestrus, whereas oxytocin (OT) binding rose more than 10-fold within this same interval. Treatment of ovariectomized rats with E2 lead to a significant increase in both OTR mRNA levels (4.4-fold) and OT binding (< 6-fold). Cotreatment with P4 strongly reduced OT binding by 75% (P < 0.01) but did not significantly affect the E2-induced rise in OTR mRNA (11% decrease, P > 0.1). Our data suggest that the increased expression of OT binding sites observed at the onset of labor and at proestrus is mediated, at least in part, by an E2-induced up-regulation of OTR gene expression. However, it also appears that OTR mRNA levels are not the sole determinants of uterine OT binding. Specifically, P4-mediated OTR down-regulation cannot be explained by an effect on OTR mRNA accumulation and may involve novel mechanisms acting at translational or posttranslational levels.