Gene regulation in eukaryotes

SIGNIFICANCE: A gene is a segment of DNA that serves as the basic unit of inheritance. To be expressed, a gene must be transcribed to make RNA, which may in turn be translated into protein. Gene regulation occurs at various phases of this complex process. For eukaryotes, this primarily pertains to the selective expression of particular proteins during development or in specific cell types.

Introduction

“Gene expression” is most commonly used to refer to transcription of genes into RNA and subsequently, translation of many of these RNAs into the proteins that carry out myriad biochemical activities. In eukaryotes, transcription occurs in the nucleus and translation in the cytoplasm. Each step in is a potential target for regulation, and abnormalities in gene regulation are associated with disease. Historically, most gene regulation has been thought to occur at the level of transcriptional initiation, but there is increasing evidence for regulation at other levels.

Nuclear RNA Polymerases and Promoters

Nuclear RNA polymerases are a group of multisubunit proteins with intrinsic enzymatic activity that share the responsibility for transcribing eukaryotic genes. I transcribes genes encoding ribosomal RNA (rRNA), RNA polymerase II transcribes protein-coding genes and some small nuclear RNA genes, and RNA polymerase III transcribes genes encoding transfer RNA (tRNA), the 5S rRNA, and some small nuclear RNAs. Additional nuclear RNA polymerases have been described but are not as well characterized. Mitochondria and chloroplasts have RNA polymerases that transcribe their DNA. These are similar to prokaryotic versions and are not discussed here.

DNA sequences known as promoters serve to position the RNA polymerases at transcriptional start sites. The RNA polymerases do not bind promoters directly. Instead, proteins called transcription factors bind to specific sequences in promoters, and the RNA polymerases bind to their cognate transcription factors. Core sequences are those recognized by a set of basal transcription factors, defined as those transcription factors required for initiation of a basal level of transcription. Activated or repressed transcription is measured with respect to this basal level. Promoters for RNA polymerases I and III have limited variability and are recognized by a finite set of ubiquitous transcription factors. In contrast, promoters for RNA polymerase II show significant diversity, and the number of transcription factors involved in positioning the polymerase is huge. Many of the promoter-binding transcription factors for RNA polymerase II are ubiquitous and mediate basal transcription, while others are gene-, cell type- or developmentally specific and involved in activation or repression of transcription.

For RNA polymerases I and III, regulation is generally global and involves a repression of transcription. For RNA polymerase II, regulation is gene-specific, which allows selective regulation of each of thousands of protein-coding genes. RNA polymerase II promoters function only at very low efficiency with the basal transcription factors, and activation is the common mode of regulation. This overview will focus on regulation of protein-coding genes.

Basal Transcription by RNA Polymerase II

RNA polymerase II promoters are modular. The core promoter, which directs transcription by the basal transcription apparatus, typically extends about thirty-five base pairs upstream or downstream of the transcriptional start site. Core promoters can vary considerably, and there are no universal core promoter elements. Common core promoter elements include the TATA-box, an AT-rich sequence that may be located about twenty-five base pairs upstream of the transcriptional start, and the region immediately surrounding the start site, known as the initiator. The downstream promoter element, DPE, may be found about thirty base pairs downstream of the transcriptional start, mainly in genes that do not have a TATA-box. The strength of a given promoter, as defined by the level of basal transcription, depends on which combination of promoter elements is present and on their respective sequences.

The core promoter elements are recognized by basal transcription factors that for RNA polymerase II are named TFIIX, where X is a letter that identifies the individual factor. For example, the TATA-box is bound by the TATA-binding protein, which is a subunit of the known as TFIID. A subset of TATA-boxes features a sequence immediately upstream that serves as a recognition site for TFIIB. TFIIB, in turn, recruits the RNA polymerase.

Regulated Transcription by RNA Polymerase II

In addition to its role in basal transcription, the core RNA polymerase II promoter contributes to regulation of transcription. Additional DNA elements called enhancers function to activate transcription from basal levels; conversely, repressors are DNA elements that function to repress transcription. Enhancers and repressors may be located on either side of the gene, up to several thousand base pairs from the core promoter and the transcriptional start site. Enhancers, repressors, and the core promoter sites involved in regulated transcription are recognized by transcription factors that mediate changes in transcriptional activity. Transcription factors show great variability in terms of cell type and gene specificity, allowing for unique regulation of individual genes. Activators are better characterized than repressors, and are modular, containing both a DNA-binding domain and an activation domain. One mode for regulation of transcription factors is phosphorylation in response to an extracellular signal. Some activators function by directly interacting with components of the transcription apparatus to stimulate transcription.

Mechanisms exist to ensure that only certain gene(s) are the target of a given enhancer. DNA insulators are sequences that prevent activation of nonassociated genes by a given enhancer. Interestingly, the insulator function is position-specific, unlike the function. Insulators are thought to function through specific insulator-binding factors.

The RNA polymerase and cognate transcription factors must have access to a given gene to accomplish transcriptional initiation. This access appears to be regulated by subnuclear localization and DNA structure. Recent work suggests that RNA polymerases are localized to discrete areas of the nucleus, termed “transcription factories,” and genes must move to these areas to be expressed. It has also been proposed that protein-coding genes localize to nuclear pores when expressed so that their products can be more readily exported to the cytoplasm for translation. This gene-gating hypothesis has received recent experimental support. Additionally, DNA sequences are normally packaged into highly organized and compacted nucleoprotein structures known as chromatin. This packaging can occlude protein-binding sites, interfering with binding of transcription factors. Chromatin packaging varies with cell cycle, cell type, and regulatory signals. Many activators function by recruiting protein complexes that remodel to increase DNA accessibility. Efficient transcription may also depend on specific elongation factors that travel with the RNA polymerase to destabilize chromatin structure.

Post-transcriptional Control

Nascent protein-coding transcripts, or pre-messenger RNAs (pre-mRNAs) are subject to several types of post-transcriptional processing in the nucleus. Intervening sequences (introns) are removed by splicing, a “cap” structure is added to the 5′ end, and a polyadenosine (poly-A) tail is added to the 3′ end, following cleavage of the transcript. Although historically referred to as post-transcriptional events, this processing occurs during, not after, transcription. The largest RNA polymerase II subunit has a carboxyl-terminal domain that serves to recruit proteins involved in mRNA splicing, polyadenylation, and capping, thus securing a tight association between these processes.

Capping and polyadenylation affect both stability of the mRNA and the efficiency of translation. Since most intracellular RNA degradation is in the form of nuclease-mediated degradation from either end, protecting the ends by cap-binding proteins and polyA-binding proteins, respectively, prevents degradation. Short-lived mRNAs often contain elements within the region downstream of the stop that explicitly recruit complexes that degrade the RNA. In general, genes that encode “housekeeping” proteins produce mRNAs with long half-lives, whereas genes whose expression must be rapidly controlled tend to generate mRNAs with short half-lives.

Additional protein diversity and regulation is generated by alternative splicing, a process whereby different combinations of coding sequences, or exons, are incorporated into the final spliced mRNA product. In this fashion, multiple versions of a protein may be made from a single gene.

Following transcription and nuclear processing, mRNAs are transported to the cytoplasm for translation. The mRNA sequence affects the efficiency with which it is translated. For instance, folding of the mRNA region upstream of the start codon can interfere with binding of the ribosome, and the sequence adjacent to the start codon affects the efficiency of translation initiation. Nucleotide sequences in the untranslated regions of mRNA are also recognized by specific proteins that may anchor the mRNA to specific cellular structures to ensure their translation and accumulation at the appropriate locations.

The RNA interference (RNAi) pathway has emerged recently as an important mechanism for negative regulation of gene expression at the RNA level. In plants, RNAi has been suggested to play an important role in resistance to pathogens, particularly viruses. This pathway utilizes the RNA-induced silencing complex (RISC) to silence specific genes through selective degradation of cytoplasmic mRNAs. Long cytoplasmic double-stranded RNA is thought to be cleaved by an identified as Dicer to form double-stranded short interfering RNAs (siRNAs) approximately twenty-two base pairs long. The siRNAs are incorporated to RISC, which separates the strands and targets the corresponding cellular mRNA for degradation by an endonuclease called Slicer (originally identified as Ago2). Interestingly, endogenous siRNAs have been identified, and other small endogenous RNAs such as microRNAs (miRNAs) may cause selective mRNA degradation through similar pathways. On the basis of genome analysis, animal cells have the potential to synthesize hundreds of different miRNAs.

Experimental Manipulation of Gene Expression

Overexpression of a gene, either mutated or in its native form, can be achieved experimentally in multiple ways, for example when exogenous DNA encoding the desired gene is introduced to the cell nucleus. Historically, it was easier to increase than it was to reduce expression of specific genes. However, powerful techniques are now available for reducing and even silencing gene expression. These approaches include recombination, antisense technology, and RNAi. Homologous recombination between a chromosomal and an introduced, manipulated copy of a gene can be used to silence (knockout) the gene, though this approach is labor-intensive. Antisense technology relies on specific between complementary single-stranded oligonucleotides and mRNA to prevent translation of the mRNA. This technique requires integration and expression of DNA encoding the appropriate sequences or introduction of specific single-stranded oligonucleotides. Finally, the RNAi pathway can be exploited by introducing synthetic double-stranded siRNAs, resulting in of the targeted gene. This approach has evolved into a powerful tool for probing gene activity and developing gene-silencing therapeutics.

Each eukaryotic cell contains the same tens of thousands of genes, so cell specialization relies on selective regulation of gene expression. The normal mechanisms of gene regulation are elegant and complex, ranging from transcriptional to translational control. Understanding normal gene regulation can reveal how dysregulation contributes to disease.

Key Terms

• antisense technologyuse of antisense oligonucleotides or nucleic acids that base pair with mRNA to prevent translation
• basal transcription factorprotein that is required for initiation of transcription at all promoters
• chromatin remodelingany event that changes the nuclease sensitivity (DNA accessibility) of chromatin
• core promotersDNA elements that direct initiation of transcription by the basal RNA polymerase machinery
• enhancera DNA element that serves to enhance transcriptional activity above basal levels
• insulatora non-position-specific DNA element that, when placed between an enhancer and a gene’s promoter, prevents activation of that particular gene
• repressora non-position-specific DNA element that serves to repress transcriptional activity below basal levels
• RNA interference (RNAi)a pathway that silences specific genes through selective degradation of RNAs
• short interfering RNA (siRNA)short, endogenous or exogenous double-stranded RNA containing specific gene sequences
• transcription factora protein that is involved in initiation of transcription but is not part of the RNA polymerase

Bibliography

"Can Genes be Turned On and Off in Cells?" MedlinePlus, 28 Mar. 2021, medlineplus.gov/genetics/understanding/howgeneswork/geneonoff/. Accessed 5 Sept. 2024.

Carthew, Richard W., and Erik J. Sontheimer. “Origins and Mechanisms of miRNAs and siRNAs.” Cell, vol. 136, no. 4, 2009, pp. 642–55.

Lewin, Benjamin, et al. Lewin's Genes XI. 11th ed., Jones, 2014.

Micklos, David A., et al. Genome Science: A Practical and Conceptual Introduction to Molecular Genetic Analysis in Eukaryotes. Cold Spring Harbor Laboratory, 2013.

Schneider, Robert, and Rudolf Grosschedl. “Dynamics and Interplay of Nuclear Architecture, Genome Organization, and Gene Expression.” Genes and Development, vol. 21, 2007, pp. 3027–43.

Wang, Zefeng, and Christopher B. Burge. “Splicing Regulation: From a Parts List of Regulatory Elements to an Integrated Splicing Code.” RNA, vol. 14, no. 5, 2008, pp. 802–13.

Watson, James D., et al. Molecular Biology of the Gene. 7th ed., Pearson, 2014.