Internal ribosome entry site (IRES) RNAs are important regulators of gene expression, but their diverse molecular mechanisms remain partially understood. The HIV-1 gag transcript leader contains an IRES that may be a good model for understanding the function of many other IRESs. We investigated the possibility that this IRES’ function is linked to both the structure of the RNA and its cellular environment. We find that in the context of a bicistronic reporter construct, HIV-1 gag IRES’ activity is cell type-specific, with higher activity in T-cell culture systems that model the natural target cells for HIV-1 infection. This finding underscores how an IRES may be fine tuned to function in certain cells, perhaps owing to cell type-specific protein factors. Using RNA probing and mutagenesis, we demonstrate that the HIV-1 gag IRES does not use pre-folded RNA structure to drive function, a finding that gives insight into how conformationally dynamic IRESs operate. Furthermore, we find that a common exon drives IRES activity in a diverse set of alternatively spliced transcripts. We propose a mechanism in which a structurally plastic RNA element confers the ability to initiate translation internally, and activity from this common element is modulated by 3′ nucleotides added by alternative splicing.

With the emergence of multi-drug resistant (MDR) and extensively drug resistant (XDR) Mycobacterium tuberculosis (Mtb), new classes of anti-mycobacterial agents with very different modes of action compared to classical antibiotics, are urgently needed. In this study, a series of 26-residue, amphipathic α-helical antimicrobial peptides consisting of all D-amino acid residues and synthetic human L-LL37 (L-enantiomer) and D-LL37 (D-enantiomer) were investigated against M. tuberculosis susceptible strain (H37Rv) and a clinical multi-drug resistant strain (Vertulo). Minimal inhibitory concentrations (MICs) were determined through a peptide killing assay. D5, the most active analog against M. tuberculosis had a MIC value of 11.2 μM (35.2 μg/ml) against H37Rv strain and 15.6 μM (49 μg/ml) against the MDR strain. Peptide D1 had similar activity as D5 against the MDR strain (57 μg/mL), a 9-fold improvement in hemolytic activity and a 7.4-fold better therapeutic index compared to D5. Surprisingly, LL37 enantiomers showed little to no activity compared to the de-novo designed α-helical antimicrobial peptides.

Alternative splicing of the human immunodeficiency virus 1 (HIV-1) RNA transcripts produces mRNAs encoding nine different viral proteins. The leader of each contains a common non-coding exon at the 5' end. Previous studies showed that the leaders from the common exon-containing transcripts gag, nef, vif, vpr and vpu can direct protein synthesis through internal ribosome entry sites (IRESs) with varying efficiencies. Here we explored whether the common exon acts as an IRES element in the context of all the 5' leaders or if each harbors a distinct IRES. We also explored the relationship between the IRESs and initiation codon selection. We find that the common exon adopts a similar conformation in every leader we explored and that the sequence and structure is required for IRES activity. We also find that each leader uses a scanning mechanism for start codon identification. Together, our data point to a model in which the common exon on HIV-1 transcripts acts as the ribosome landing pad, recruiting preinitiation complexes upstream of the initiation codon, followed by scanning to each transcript's initiator AUG.

Classical antibiotics work by inhibiting bacterial cell wall synthesis, protein synthesis, DNA (deoxyribonucleic acid) replication, or by modifying metabolism. Bacteria can resist antibiotics as a result of chromosomal changes (mutation or inductive expression of a latent chromosomal gene) or the exchange of genetic material via plasmids and transposons. As this chapter will discuss, antibiotics are rendered inactive by three major mechanisms: (1) antibiotic inactivation by destruction or modification, (2) prevention of access of the antibiotic to the target, and (3) alteration of the target site of the antibiotic. Bacteria have a remarkable ability to overcome each new reagent synthesized as a potential classical antibiotic. Consequently, the current priority is the development of alternative drugs and/or the isolation of native molecules that would allow the consistent and proper control of pathogen‐caused diseases.