The Gin function of bacteriophage Mu catalyzes inversion of the G DNA segment, thus switching the host range of Mu phage particles. This site-specific recombination event takes place between inverted repeat sequences (IR) that border the G segment. Sequences in the Mu beta region extending approximately from position 118 to 178 are essential for efficient inversion. In cis this region, termed sis, stimulates inversion about 15-fold. Neither the relative orientation of sis with respect to the IR sequences nor the distance to IR substantially influences the stimulatory effect. For full activity purified Gin protein must be supplemented with crude host factor from E. coli K12. We suggest that, in addition to Gin, a DNA-binding host protein is required for efficient G inversion.

No abstract

Ribosomal protein L5 forms a small, extraribosomal complex with 5 S ribosomal RNA, referred to as the 5 S ribonucleoprotein complex, which shuttles between nucleus and cytoplasm in Xenopus oocytes. Mapping elements in L5 that mediate nuclear protein import defines three separate such activities (L5-nuclear localization sequence (NLS)-1, -2, and -3), which are functional in both oocytes and somatic cells. RNA binding activity involves N-terminal as well as C-terminal elements of L5. In contrast to the full-length protein, none of the individual NLSs carrying L5 fragments are able to allow for the predominating accumulation in the nucleoli that is observed with the full-length protein. The separate L5-NLSs differ in respect to two activities. Firstly, only L5-NLS-1 and -3, not L5-NLS-2, are capable of promoting the nuclear transfer of a heterologous, covalently attached ribonucleoprotein complex. Secondly, only L5-NLS-1 is able to bind strongly to a variety of different import receptors; those that recognize L5-NLS-2 and -3 have yet to be identified.Large quantities of 5 S ribosomal RNA are synthesized in excess over other ribosomal components and stored in the cytoplasm during early stages of oogenesis in Xenopus (1). 5 S rRNA storage occurs in the context of small, nonribosomal ribonucleoprotein complexes (RNPs), 1 primarily in association with transcription factor IIIA (7 S RNP) (2-4), but also as part of a larger RNP, the 42 S RNP, which contains tRNA and the two proteins, p48 and p43, in addition to 5 S rRNA (5-7). Later in oogenesis, synthesis of the primary 5 S rRNA binding ribosomal protein L5 increases (8), resulting in the formation of the preribosomal 5 S RNP (9). Binding of newly synthesized 5 S rRNA to either L5 or TFIIIA is necessary for nuclear export of the corresponding RNPs in Xenopus oocytes (10). During later stages of oogenesis, concurrent with increased assembly of ribosomal subunits, the cytoplasmically stored 5 S rRNA is reimported into the nucleus and subsequently utilized for ribosome assembly in the nucleoli. This reimport of 5 S rRNA occurs in a complex with L5. In contrast, the complex with TFIIIA remains sequestered in the cytoplasm, which is due to masking of the NLS function in TFIIIA by the associated 5 S rRNA (11-14).The nucleocytoplasmic transport of proteins and RNA proceeds though the nuclear pore complex, a large, multiprotein complex embedded in the nuclear membrane. Ions, small metabolites, and small proteins can passively diffuse through the aqueous channel of the nuclear pore complex, whereas larger molecules are actively transported through the gated channel of the nuclear pore. Nucleocytoplasmic transport of macromolecules is a signal-mediated process and requires various transport factors, some of which interact with the nuclear pore complex (15-19). The directionality of the translocation process is controlled by the RanGTPase system (20 -22). RanGTP allows for the dissociation of import complexes in the nucleus and is also required for the formation of export complexe...

Nuclear export of newly transcribed 5S ribosomal RNA in Xenopus oocytes occurs in the context of either a complex with the ribosomal protein L5 (5S RNP) or with the transcription factor IIIA (7S RNP). Here we examine nuclear import of 5S RNA, L5 and TFIIIA. The 5S RNP shuttles between nucleus and cytoplasm and only 5S RNA variants which can bind to L5 gain access to the nucleus. The 7S RNP is retained in the cytoplasm. Only TFIIIA which is not bound to 5S RNA is imported into the nucleus. As a novel mechanism for cytoplasmic retention, we propose that RNA binding masks a nuclear localization sequence in TFIIIA. In contrast to the nuclear import of L5, import of TFIIIA is sensitive towards the nuclear localization sequence (NLS) competitor p(lys)‐BSA, suggesting that these two proteins make use of different import pathways.

C 2 H 2 -type zinc-finger modules define a unique structural motif, which is capable of forming specific complexes with both DNA and RNA. While the principles governing DNA binding have been defined in great detail, the mode of RNA recognition remains only poorly understood. In the absence of information from three-dimensional structural analysis of a zinc-finger/RNA complex, we have performed a number of biochemical studies to gain further insight into the molecular details of the interaction of 5S ribosomal RNA with the zinc-finger protein TFIIIA. Previous work had indicated that zinc finger 6 of TFIIIA contacts 5S RNA in close proximity or directly in the loop-A region (nucleotides 10Ϫ13). Permutation analysis of this sequence reveals that three of the four nucleotides are of vital importance for RNA recognition. Exchange of unusual and therefore characteristic aromatic residues in finger 6 against aliphatic or other aromatic amino acids reveals that the aromatic character of tryptophan 177 is essential for RNA recognition. Association with helix V in 5S RNA appears to involve specific contacts with the phosphate backbone, as evidenced by ethylation-interference assays. Introduction of multiple internal and 3′-terminal as well as 5′-terminal deletions accompanied by stabilizing sequence substitutions defines a minimal RNA fragment that is sufficient for TFIIIA binding. This RNA molecule includes a truncated/ mutated helix I, helix II and helix V, as well as structurally intact loops A and E. Permutation analysis of the loop-E region emphasizes its importance for TFIIIA recognition.Keywords : TFIIIA; 5S RNA; RNA-protein interaction; zinc finger.Of the many zinc-finger proteins that exist, only few bind to RNA [1Ϫ7]. TFIIIA from Xenopus laevis, the first non-viral eukaryotic transcription factor described, which is specific for the transcription of 5S RNA genes by RNA polymerase III [1,8,9], is one of these. Besides binding to the internal control region of the 5S RNA gene, this protein is part of a cytoplasmic 5S RNA storage particle, which sediments at 7S and acts as a transporter of 5S RNA during nucleocytoplasmic transfer [10]. The zinc-finger region mediates binding to both DNA and RNA. The nine zinc fingers of TFIIIA differ in their importance for binding to DNA or RNA; zinc fingers 1, 2, and 3 are sufficient for specific binding to the 5S RNA gene [11Ϫ15]; fingers 5, 7, 8, and 9 also appear to be involved in the formation of basespecific contacts within the major groove of the DNA [16]; in contrast, fingers 4 and 6 are thought to bridge the minor groove, without participating in DNA sequence recognition [17]. Wuttke et al. [18] have presented a solution structure of zinc fingers 1, 2, and 3, with the corresponding DNA recognition element, in principle confirming the model derived from previous studies with zinc fingers contacting more than three base pairs. Equivalent structural information for the RNA/zinc finger complex is not yet available.The RNA binding activity of TFIIIA has been investigated by me...