The hepatitis C virus (HCV) nonstructural protein 5B (NS5B) is believed to be the central catalytic enzyme responsible for HCV replication but there are many unanswered questions about how its activity is controlled. In this study we reveal that two other HCV proteins, NS3 (a protease/helicase) and NS4B (a hydrophobic protein of unknown function), physically and functionally interact with the NS5B polymerase. We describe a new procedure for generating highly pure NS4B, and use this protein in biochemical studies together with NS5B and NS3. To study the functional effects of the protein-protein interactions, we have developed an in vitro replication assay using the natural noncoding 3 regions of the respective positive ((؉)-3 -untranslated region) and negative ((؊)-3 -terminal region) RNA strands of the HCV genome. Our studies show that NS3 dramatically modulates template recognition by NS5B and changes the synthetic products generated by this enzyme. The use of an NTPase-deficient mutant form of NS3 demonstrates that the NTPase activity (and thus helicase activity) of this protein is specifically required for these effects. Moreover, NS4B is found to be a negative regulator of the NS3-NS5B replication complex. Overall, these results reveal that NS3, NS4B, and NS5B can interact to form a regulatory complex that could feature in the process of HCV replication.Hepatitis C virus (HCV) 1 is a major pathogen of parenterally transmitted non-A, non-B hepatitis (1) and often causes the development of malignant chronic disease, including liver cirrhosis and hepatocellular carcinoma (2). With nearly 3% of the population of the world infected with HCV and no protective vaccine available at present, this disease has emerged as a serious global health problem since the virus was first identified (3, 4). HCV is a positive-stranded RNA virus with a genome of ϳ9400 bp. This genomic RNA initially directs the synthesis of at least 10 structural and nonstructural viral proteins (5). Following that, it is utilized by the viral RNA-dependent RNA polymerase (the nonstructural protein 5B; NS5B) as template to generate a complementary negative-stranded RNA. Once synthesized, the negative strands are transcribed into new molecules of positive-stranded genomic RNA, which in turn provide additional templates for viral protein synthesis as well as genomic RNA for the production of progeny virus (5, 6). However, the molecular events that mediate this process remain largely unclear.Several attempts to dissect the mechanistic details of the viral replication cycle have been reported to date. The focal point of such investigations has been NS5B, which possesses an RNA-dependent RNA polymerase (RdRp) activity and is believed to be the key enzyme catalyzing HCV RNA synthesis (7-14). Its crystal structure reveals that it contains the classical finger, palm, and thumb subdomains of the polymerases with the unique feature of a more fully enclosed active site tunnel (15)(16)(17), and a recent report by Bressanelli and colleagues (18) has provided add...

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The experiments here reported demonstrate that the main non-coding region of rat mitochondrial DNA is symmetrically transcribed. We have identified stable heavy and light transcripts, whose pattern is rather complex, in the D-loop region of rat mitochondrial DNA. Their relative concentrations have been determined. We detected heavy transcripts which encompass the whole D-loop and more abundant heavy RNA species which we interpreted as transcripts terminating downstream of the 3' end of the last coded gene (Thr-tRNA). The processed heavy RNA species contain polyA, suggesting a strict association between cleavage and polyadenylation. The pattern of light transcripts shows a long RNA, which, starting from the light strand promoter, covers the whole segment, and shorter RNA species which seems to be actively processed at the level of the conserved sequence boxes, probably acting as primers. The symmetric transcription of the D-loop containing region of rat mitochondrial DNA, and in particular the presence of stable transcripts complementary to the putative RNA primers, suggest that mechanisms mediated by interaction between complementary transcripts (antisense RNAs) might play a role in the regulation of mitochondrial DNA replication and expression.

In the process of protein synthesis, the small (40S) subunit of the eukaryotic ribosome is recruited to the capped 5 end of the mRNA, from which point it scans along the 5 untranslated region in search of a start codon. However, the 40S subunit alone is not capable of functional association with cellular mRNA species; it has to be prepared for the recruitment and scanning steps by interactions with a group of eukaryotic initiation factors (eIFs). In budding yeast, an important subset of these factors (1, 2, 3, and 5) can form a multifactor complex (MFC). Here, we describe cryo-EM reconstructions of the 40S subunit, of the MFC, and of 40S complexes with MFC factors plus eIF1A. These studies reveal the positioning of the core MFC on the 40S subunit, and show how eIF-binding induces mobility in the head and platform and reconfigures the head-platform-body relationship. This is expected to increase the accessibility of the mRNA channel, thus enabling the 40S subunit to convert to a recruitment-competent state.posttranscriptional gene expression ͉ protein synthesis ͉ ribosome structure E lucidation of the mechanisms underlying ribosome function and protein synthesis remains one of the major challenges of molecular biology. Recent progress in structural analysis of bacterial ribosomes has provided insight into the likely modes of action of core functional centers, including those for decoding and peptidyl transferase, and the tRNA-binding sites (1). Analogous core structural features are clearly shared by the eukaryotic counterpart, but there is much less structural and mechanistic information available that is specific to the eukaryotic ribosome. This limits our understanding of the process of translation initiation, the step where major differences between the prokaryotic and eukaryotic systems are evident (2).The small ribosomal subunit in both prokaryotes (30S) and eukaryotes (40S) is responsible for controlling base pairing between the tRNA anticodon and each mRNA codon during protein synthesis. However, unlike its prokaryotic (30S) counterpart, the eukaryotic 40S subunit does not locate directly to the position of the mRNA AUG codon where protein synthesis begins. Instead, recruitment onto cellular mRNAs generally occurs via the capped 5Ј end. Because the AUG start codon can be located many hundreds of nucleotides downstream, the 40S subunit then has to translocate to reach the initiation site (3) [see supporting information (SI) Fig. 5]. During this processive, sequence-independent scanning phase, the 40S subunit manifests some characteristics that appear to be similar to those of a molecular motor (2, 4).The eukaryotic 40S subunit alone is incapable of stable recruitment onto the capped 5Ј ends of cellular mRNA molecules. Its role in translation initiation depends on a large number of eukaryotic initiation factors [the eIFs (5)]. According to the current classification, 11 distinct eIFs (including eIF2B, a guanine nucleotide exchange factor) are involved in (steady-state) translation initiation. There has bee...

We have identified new transcripts in the region surrounding the L-strand replication origin (Ori L) of rat liver mitoehondrial DNA. In particular. we have detected previously unidentified intermediates of RNA processing on both the heavy and the light strands, such as precursors ol'the ND2 mRNA plus the Trp-tRNA and precursors of the tRNAs clustered in the Ori L region. This indicates that the mechanism of RNA processing in mitoehondria proceeds step-wise producing a variety of precursors of the instate forms. The other striking finding is the detection of antisense RNA species in the region of L-strand replication. Since a variety ofantisense transcripts were also found in the D-loop region of rat mitochondrial DNA, we suggest that they Hight play a regulatory role in the replication and expression ~f the mitochondrial genome.

bstract We have characterized the transcriptional pattern of the rat mitochondrial ND6containing region in vivo. We have identified a stable nyadenylated RNA species complementary for the full length of the ND6 mRNA. The analysis of the ND5 region has revealed the presence of I antisense RNA only at its 3' end. The presence of these stable antisense species complementary to structural genes is intriguing and suggests a rssible regulatory function. The quantitative analyses have demonstrated that the H transcripts, both codogenic and non-codogenic, are more stable an the L transcriptswe have defined the 5' end of the ND6 mRNA at the level of the ATG downstream of the tRNAo'". The mapping of the ND1 end has demonstrated that GTG is the first codon of the mRNA. Our findings suggest that the post-transcriptional mechanisms involved in the pression of the mt genome are much more numerous and complex than those already described in the literature.

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We have analyzed the tRNALy', ATPase8, ATPase6, CO111 region of mitochondrial DNA in several human tissues. Beside the mature tRNA"', ATPaseS and ATPase6 common mRNA, and CO111 mRNA, we have characterized two new transcripts, called RNA 20 and RNA 21. The RNA 20 is a precursor species which contains the tRNALyS plus the ATPasel and ATPase6 common mRNA; the RNA 21 is an RNA species shorter than the ATPase8 and ATPase6 common mRNA. The relative concentration of the mature with respect to that of the new species proved different in the various tissues. These findings provide new insights into the mitochondrial transcription mechanism opening the question of a possibly regulatory role of the processing on the expression of the mitochondrial genome.