Gene expression relies on messenger RNA (mRNA) to carry the genetic blueprint from the DNA to the ribosomes, where proteins are synthesized. In most eukaryotic organisms, this process is governed by the rule that one gene sequence on the mRNA codes for one protein. The term “bicistronic” describes an exception, referring to a single mRNA molecule that contains the genetic instructions for two distinct proteins. This arrangement allows for the coordinated production of two separate proteins from a single transcript, providing a powerful mechanism for gene regulation.
Monocistronic Versus Bicistronic Messages
Eukaryotic cells primarily use “cap-dependent initiation,” resulting in monocistronic messages. A monocistronic mRNA contains a single Open Reading Frame (ORF), which is translated into one protein. Translation begins when the ribosome recognizes the 5′ cap, a chemically modified guanine nucleotide located at the beginning of the mRNA strand.
The ribosome complex binds to the 5′ cap and scans the mRNA until it encounters the first start codon, typically AUG, initiating protein synthesis. Once translation is complete, the ribosome dissociates. This mechanism ensures that only the first ORF is translated, meaning downstream coding sequences are normally ignored.
A bicistronic mRNA contains two distinct ORFs separated by non-coding RNA. Since the ribosome releases the mRNA after translating the first ORF, the second ORF cannot be translated by the standard cap-dependent mechanism. To overcome this roadblock, the bicistronic message must contain a specialized element allowing the ribosome to initiate translation internally. This element is the Internal Ribosome Entry Site (IRES), the defining feature of a bicistronic message.
The Role of the Internal Ribosome Entry Site
The Internal Ribosome Entry Site (IRES) is an intricate RNA structure that acts as a translational bypass, allowing the ribosome to initiate protein synthesis without relying on the 5′ cap. The IRES is positioned in the non-coding space between the two ORFs on the bicistronic mRNA. Structurally, the IRES is defined by a complex three-dimensional folding pattern, which is unique to each type.
This folded RNA sequence recruits the small 40S ribosomal subunit directly to an internal position on the mRNA, skipping the typical 5′ cap recognition and scanning steps. Different IRES elements have varying requirements for eukaryotic initiation factors (eIFs). For example, some viral IRES structures are highly efficient and can bind the ribosome with few or no initiation factors.
The IRES serves as a second, independent landing pad for the ribosome, ensuring the second ORF is translated after the first protein has been produced. This cap-independent translation is useful for cells under stress or during viral infection, where the normal cap-dependent translation machinery may be inhibited, allowing IRES-containing messages to be preferentially translated.
Natural Sources of Bicistronic Expression
Bicistronic expression, driven by IRES elements, is most commonly observed in the life cycles of various viruses, such as picornaviruses like Poliovirus and Encephalomyocarditis virus (EMCV). Viruses exploit the IRES mechanism as a survival strategy, ensuring the rapid production of their own proteins within the host cell. The viral genome is often a single, long RNA molecule that functions as a bicistronic or polycistronic message.
Upon infecting a host cell, many viruses employ proteases to cleave the host’s translation initiation factors, specifically targeting the factor that binds to the 5′ cap. This action shuts down the cell’s cap-dependent protein synthesis, stopping the production of host proteins. However, the viral mRNA, which contains an IRES, remains active, allowing the virus to commandeer the translational machinery for replication.
While viral IRES elements are the most studied, a smaller number of cellular genes in higher organisms also contain them. These cellular IRESs are often found in mRNAs that encode proteins involved in stress responses, cell cycle control, or apoptosis. During cellular stress, such as nutrient deprivation or hypoxia, when cap-dependent translation is suppressed, these cellular IRESs ensure that necessary proteins are still synthesized.
Applications in Gene Editing and Therapy
The efficiency and coordinated expression offered by bicistronic systems make them a powerful tool in biotechnology and gene therapy. Scientists use IRES elements to engineer genetic constructs that ensure the co-expression of two different genes from a single promoter. This is accomplished by placing the IRES sequence between the two desired coding sequences within a single vector.
A frequent application is linking a therapeutic protein—the gene of interest—with a reporter gene, such as Green Fluorescent Protein (GFP) or a drug resistance marker. Since both genes are on the same transcript, the presence of the reporter protein confirms that the therapeutic gene is also successfully expressed. This allows researchers to easily track and quantify cells that have taken up the genetic material.
Bicistronic vectors are extensively used in creating stable cell lines and in gene therapy applications, including those using Adeno-Associated Virus (AAV) vectors. AAV vectors have a limited cargo capacity, and the IRES allows for the packaging of two genes into the restricted space of a single vector. This dual expression is valuable for complex treatments requiring the simultaneous delivery of multiple genes, such as combining a therapeutic factor with a selective marker.