In addition to the cap-dependent mechanism, eukaryotic initiation of translation can occur by a cap-independent mechanism which directs ribosomes to defined start codons enabled by internal ribosome entry site (IRES) elements. IRES elements from poliovirus and encephalomyocarditis virus are often used to construct bi- or oligocistronic expression vectors to co-express various genes from one mRNA. We found that while cap-dependent translation initiation from bicistronic mRNAs remains comparable to monocistronic expression, internal initiation mediated by these viral IRESs is often very inefficient. Expression of bicistronic expression vectors containing the hepatitis B virus core antigen (HBcAg) together with various cytokines in the second cistron of bicistronic mRNAs gave rise to very low levels of the tested cytokines. On the other hand, the HBcAg was well expressed when positioned in the second cistron. This suggests that the arrangement of cistrons in a bicistronic setting is crucial for IRES-dependent translation of the second cistron. A systematic examination of expression of reporter cistrons from bicistronic mRNAs with respect to position was carried out. Using the dual luciferase assay system we show that the composition of reading frames on a bicistronic mRNA and the order in which they are arranged define the strength of IRES-dependent translation. Although the cellular environment and the nature of the IRES element influence translation strength the dominant determinant is the nature and the arrangement of cistrons on the mRNA.

The ubiquitous transcription factor NRF (NF-B repressing factor) is a constitutive transcriptional silencer of the multifunctional cytokine interferon-␤. NRF mRNA contains a long 5 untranslated region (5UTR) predicted to fold into a strong secondary structure. The presence of stable hairpins is known to be incompatible with efficient translation by ribosomal scanning. Using dicistronic reporter gene constructs, we show that the NRF 5UTR acts as an internal ribosome entry site (IRES) which directs ribosomes to the downstream start codon by a cap-independent mechanism. The relative activity of this IRES in various cell lines is at least 30-fold higher than that of picornaviral IRESs. The NRF 5UTR also functions as a translational enhancer in the context of monocistronic mRNAs. Our results indicate that the NRF 5UTR contains a highly potent IRES, which may allow for an alternate mode of translation under physiological conditions in which cap-dependent translation is inhibited.

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We have used a phage-display-based system to investigate the effect of simultaneous substitutions within the C5a effector domain. Two different libraries were constructed. In library I, known binding positions 67, 68, 72 and 74 of human complement C5a (hC5a) and in library II, positions 69Ϫ73 of hC5a without C-terminal Arg74 (des-Arg74-C5a) were randomly mutated. In more than 80% (position 72) or 90% (positions 68 and 74) of all cases, the original residues of hC5a were selected from library I, demonstrating that the phage system can be used to define binding points within the C5a molecule. Surprisingly, in more than 90% of all clones, a Phe residue was enriched at position 67 instead of the original His residue which, however, did not affect the binding affinity or the signalling activity. In library II, Leu was preferentially selected at positions 70Ϫ72 and Tyr at position 73, while no enrichment of an individual amino acid was observed at position 69. Mutants with (a) Leu in positions 71 and 72 (b) Ser or Leu in position 70 and (c) Arg or Tyr in position 73, showed a 4Ϫ10-fold higher binding affinity as compared to des-Arg74-[Ala27, Phe67]C5a. The binding affinity was indistinguishable from that of hC5a. In consequence, not only position 72 but also positions 70, 71 and 73 are able to interact with the C5a receptor, whereas position 69 is not. Intriguingly, one mutant with a high binding affinity but without signalling activity was selected. Thus, random mutagenesis of phage-displayed C5a was proven to be a powerful strategy to define receptor-binding points and to select C5aR antagonists based on the structure of the natural ligand.Keywords : complement 5a; complement 5a receptor; filamentous phage ; phage display; cell panning.During complement activation, a panel of proinflammatory fide bonds. The flexible C-terminus points away from the core into solution. mediators is generated, one of which is the C5a anaphylatoxin. It exerts a variety of biologic activities (for review, seeThe functional activities of C5a are mediated by a specific receptor (C5aR), which belongs to the family of seven-transwhich explain its causal involvement in both acute and chronic inflammatory diseases, such as sepsis or rheumatoid arthritis (for membrane domain GTP-binding-protein (G-protein)-coupled receptors [1]. The involvement of both, the C-terminus and Nreview, see [2]). To understand the functional properties of this terminus of the C5a molecule in receptor binding and activation molecule in more detail, a series of structural studies has been has been demonstrated using C-terminal and N-terminal deletion performed. NMR spectroscopy of bovine [3] and human C5a [4] mutants [6, 7]. Singular and multiple mutations at the C-termiand fluorescence spectroscopy of human C5a (hC5a) mutants nus, the disulfide-linked core and the N-terminus revealed 3Ϫ4 Leu72 and Arg74. The importance of Arg40, however, has been E-mail: koehl.joerg@mh-hannover.de questioned in a recent study [11]. Abbreviations. ABICAP, antibody immuno column for ana...

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