Transcription initiation by archaeal RNA polymerase (RNAP) is assisted by transcription factors, TBP, TFB, and TFEα, which show sequence, structural and functional homology to eukaryotic TBP, TFIIB, and TFIIEα. Previous low-resolution cross-linking studies of transcription complexes from the hyperthermophile archaeon Pyrococcus furiosus showed that the transcription bubble DNA makes extensive contacts with TFB and TFEα. Here, contacts between TFB and TFEα surfaces and promoter DNA are mapped with amino acid-to-base precision, using a high-resolution cross-link mapping method. The non-canonical amino acid p-benzoyl phenylalanine (Bpa) was incorporated into TFB1 or TFEα at several positions, transcription complexes were formed and cross-links induced, and the positions of cross-links were mapped by primer extension. In transcription pre-initiation complexes forming a transcription bubble (PICs), the TFB1 B-reader helix and loop conserved sequences interact with the DNA transcribed (T) strand near the upstream side of the transcription bubble, while the B-linker strand conserved sequence interacts with both the transcribed and the non-transcribed (NT) strands. In a 5 nucleotide initially transcribing complex (ITC), cross-links to TFB1 changed for the B-reader but not the B-linker, suggesting scrunching of part of the T strand in the RNAP main channel. The TFEα winged helix cross-linked the NT strand at the upstream edge of the transcription bubble in the PIC. In the ITC, the NT strand changed position dramatically relative to the TFEα wing tip, indicating that the DNA NT strand can bulge out of the main channel as transcription begins and downstream DNA is pulled in. The cross-linking data were compared with structural models of the archaeal PIC and ITC, and indicate that interactions between TFB conserved motifs and DNA are conserved from archaea to humans, while archaeal TFEα contacts to DNA are apparently dissimilar from what is seen in human RNAP II initiation complexes.
MicroRNAs (miRNAs) are small, noncoding RNAs that have emerged as powerful players in the post-transcriptional regulation of gene expression. In the context of viral infections, cellular miRNAs play significant roles in modulating the complex host responses to pathogens and have essential roles in shaping viral replication. Moreover, several virus families encode viral miRNAs that manipulate the host environment and can tap into miRNA-regulated gene networks. Viral antigen expression itself can be directly miRNA regulated, and infection outcomes are substantially shaped by the miRNA activities that influence host anti-viral defense mechanisms. This review discusses common themes for both human and viral miRNAs in viral infection processes, highlighting their dual functions in both suppressing and promoting viral life cycles.
Recombinant proteins with applications in human and veterinary medicine are mainly produced in mammalian expression systems, particularly Chinese hamster ovary (CHO) cells. Although these cells enable proper folding and post-translational modifications, they often exhibit low specific productivity, which represents a critical limitation in large-scale biopharmaceutical manufacturing. Strong viral promoters, including CMV and its enhancer-enriched variant CMV+E, are commonly used to drive transgene expression. However, they are susceptible to epigenetic silencing and progressive loss of activity during prolonged culture, which can compromise yield stability. Endogenous host-cell promoters constitute a promising alternative, as they can maintain transgene expression in alignment with physiological and bioprocess regulatory mechanisms. Here, we assessed the activity of endogenous eukaryotic promoters previously identified in our laboratory from highly expressed CHO-K1 genes. The receptor-binding domain (RBD) of SARS-CoV-2 was selected as a model of a complex secretory glycoprotein domain. Our results demonstrate that the Hspa5 and Vim promoters enable sustained and high-level expression of recombinant RBD protein in CHO-K1 cells, achieving performance comparable to the enhancer-containing viral promoter CMV+E. Notably, Hspa5 and Vim are unoptimized genomic sequences lacking classical enhancers; nevertheless, their intrinsic stability and reduced susceptibility to silencing make them attractive regulatory elements for recombinant protein production. These findings highlight their potential for next-generation promoter engineering and for the development of long-term, high-yield mammalian expression systems for complex therapeutic proteins.
Proper transcription termination coupled to pre-mRNA cleavage and polyadenylation is an important step in gene expression as it generates the 3'UTR of mRNAs and defines transcription units. Defective termination (transcriptional readthrough) can suppress transcription of downstream genes by transcriptional interference but can also promote the production of chimeric transcripts, which contain the exons of upstream genes spliced to exons of downstream genes. This review will summarize current findings of chimeric splicing between exons of adjacent genes generated by transcriptional readthrough and the conditions that promote these events. Chimeric splicing can be facilitated by inactivation of factors that promote transcription termination or of specific splicing factors, particularly those involved in 3'-splice site recognition. This effect is due to the tight coupling between recognition of the terminal intron 3'-splice site and of the downstream polyadenylation sites, and its impact on transcriptional termination. Production of chimeric transcripts is also increased in cellular conditions that perturb transcription termination, such as cellular stress, treatment with the chemotherapeutic agent Imatinib or viral infections. However, not all conditions that promote transcriptional readthrough result in chimeric splicing, suggesting that the production of chimeric mRNAs requires a "Goldilocks" state of alteration of the transcriptional machinery that allows for transcriptional readthrough without a general reduction of splicing efficiency.
