Kinesin, a two-headed motor enzyme molecule, hydrolyses ATP to direct organelle transport along microtubules. As it moves along a microtubule, kinesin remains associated with, or 'tracks', microtubule protofilaments. We have prepared truncated kinesin derivatives that contain either two mechanochemical head domains or only a single head. Unlike intact kinesin and the two-headed derivatives, the one-headed enzyme frequently fails to track protofilaments, suggesting that it detaches from microtubules during movement. In this way, the one-headed kinesin derivative is similar to the motor enzyme myosin, which frequently detaches from the actin filament during movement. For myosin (which has two heads), the consequence of this detachment is that single molecules do not appear to drive continuous movement along the filament. Our observations suggest that the ability of single two-headed kinesin molecules to drive continuous movement results from a 'hand-over-hand' mechanism in which one head remains bound to the microtubule while the other detaches and moves forwards.

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Intron excision is an essential step in eukaryotic gene expression, but the molecular mechanisms by which the spliceosome accurately identifies splice sites in nuclear precursors to messenger RNAs (pre-mRNAs) are not well understood. A bimolecular assay for the second step of splicing has now revealed that exon ligation by the human spliceosome does not require covalent attachment of a 3' splice site to the branch site. Furthermore, accurate definition of the 3' splice site in this system is independent of either a covalently attached polypyrimidine tract or specific 3' exon sequences. Rather, in this system 3' splice site selection apparently occurs with a 5' --> 3' directionality.

In most eukaryotic introns the 3 splice site is defined by a surprisingly short AG consensus found a variable distance downstream of the branch site. Exactly how the spliceosome determines which AG to use, however, is not well understood. Previously we showed that when the branch site and 3 splice site AG are supplied by separate RNA molecules, there is a strong preference for use of the 5 -most AG in the 3 splice site-containing RNA. Here we show that this apparent 5 33 directionality holds even when this RNA contains four tandem repeats of a 6-nt sequence containing AG. Exactly the same pattern of 3 splice site choice was observed when the same tandem repeats were incorporated into a full-length splicing substrate. When the 3 splice site AG is supplied by a separate RNA, that RNA must be linear with an unobstructed 5 end. Similarly, the branch-containing RNA must be truncated immediately 3 to the polypyrimidine tract. A model is presented that incorporates these observations and reconciles previously proposed mechanisms for 3 splice site selection. P recise excision of introns is an essential step in eukaryotic gene expression. Most introns in the human genome (10 5 -10 6 different sequences) are removed by a single macromolecular machine, the major spliceosome. Within this machine, intron excision occurs in two steps: cleavage at the 5Ј splice site coupled with lariat formation at the branch site, followed by exon ligation at the 3Ј splice site (1, 2). However, the exact mechanisms by which these sites are recognized are not entirely understood. This is particularly true for the 3Ј splice site. In budding yeast, the only conserved sequence defining this key site is a YAG͞ trinucleotide (where Y denotes pyrimidine and ''͞'' denotes the splice site) found a variable distance downstream of the branch site. In mammals the region between the branch site and YAG͞is generally rich in pyrimidines (the polypyrimidine tract or PPT) (1,3,4). Notably, the natural 3Ј splice site in some introns is an AAG͞or GAG͞(5), with the order of preference in a test system being CAG Ϸ UAGϾAAGϾGAG (6). Thus, the sequence defining the exact site of exon ligation is apparently just AG, which could occur randomly every 16 dinucleotides. Given such odds, the spliceosome undoubtedly uses other information to distinguish the correct 3Ј splice site AG from any others nearby. Exactly what that information is, however, and how it is processed by the splicing machinery remain a matter of open debate (see ref. 7 and Discussion).To better study the mechanisms of 3Ј splice site selection by the human spliceosome, we recently developed a bimolecular (trans) assay for exon ligation in HeLa nuclear extracts (8). In this system, spliceosomes assemble and undergo the first chemical step on a 5Ј substrate consisting of a 5Ј exon and intron truncated immediately 3Ј to the PPT (Fig. 1 A). Exon ligation is then initiated by addition of a separate RNA, the 3Ј substrate, containing one or more AGs plus a downstream exon. Previously we found that the only seq...