Gene expression is primarily regulated at the pre-transcriptional level, but there are a number of mechanisms for regulation of translation as well. One well-studied animal system is the iron-sensitive RNA-binding protein, which regulates the expression of genes involved in regulating intracellular levels of iron ions. Two of these genes, ferritin, which safely sequesters iron ions inside cells, and transferrin, which transports iron from the blood into the cell, both utilize this translational regulation system in a feedback loop to respond to intracellular iron concentration, but they react in opposite ways. The key interaction is between the iron response elements (IRE), which are sequences of mRNA that form short stem-loop structures, and IRE-BP, the protein that recognizes and binds to the IREs. In the case of the ferritin gene, the IRE sequences are situated upstream of the start codon. When there is high iron, the IRE-BP is inactive, and the stem-loop structures are melted and overrun by the ribosome, allowing translation of ferritin, which is an iron-binding protein. As the iron concentration drops, the IRE-BP is activated and binds around the IRE stem-loop structures, stabilizing them and preventing the ribosome from proceeding. This prevents the production of ferritin when there is little iron to bind.
Figure 8. Pre-translational control of gene expression by iron-response protein (IRP), which binds to either the iron-response element (IRE), unless it has bound iron.
Transferrin also uses iron response elements and IRE-binding proteins, but in a very different mechanism. The IRE sequences of the transferrin gene are located downstream of the stop codon, and play no direct role in allowing or preventing translation.
However, when there is low intracellular iron and there is a need for more transferrin to bring iron into the cell, the IRE-BP is activated as in the previous case, and it binds to the IREs to stabilize the stem-loop structures. In this case; however, it prevents the 3’ poly-A tail degradation that would normally occur over time. Once the poly-A tail is degraded, the rest of the mRNA is destroyed soon thereafter. As mentioned in the transcription chapter, the longer poly-A tails are associated with greater persistence in the cytoplasm, allowing more translation before they are destroyed. The IRE-BP system in this case externally prolongs the lifetime of the mRNA when that gene product is needed in higher amounts.
Since mRNA is a single-stranded nucleic acid and thus able to bind complementary sequence, it is not too surprising to find that one of the ways that a cell can regulate translation is using another piece of RNA. Micro RNAs (miRNAs) were discovered as very short (~20 nucleotides) non-protein-coding genes in the nematode, C. elegans. Since their initial discovery (Lee et al, Cell 75: 843-54, 1993), hundreds have been found in various eukaryotes, including humans. The expression pattern of the miRNA genes is highly specific to tissue and developmental stage. Many are predicted to form stem-loop structures, and appear to hybridize to 3’-untranslated sequences of mRNA thus blocking initiation of translation on those mRNA molecules. They may also work through a mechanism similar to the siRNA discussed below, but there is clear evidence that mRNA levels are not necessarily altered by miRNA-directed translational control.
MicroRNAs are currently under investigation for their roles as either oncogenes or tumor suppressors (reviewed in Garzon et al, Ann. Rev. Med. 60: 167-79, 2009). Approximately half of known human miRNAs are located at fragile sites, breakpoints, and other regions associated with cancers (Calin et al, Proc. Nat. Acad. Sci. (USA) 101: 2999-3004, 2004). For example, miR-21 is not only upregulated in a number of tumors, its overexpression blocks apoptosis - a necessary step to allow abnormal cells to continue to live and divide rather than die out. Conversely, miR-15a is significantly depressed in some tumor cells, and overexpression can slow or stop the cell cycle, even inducing apoptosis.
Another mechanism for translational control that uses small RNA molecules is RNA interference (RNAi). This was first discovered as an experimentally induced repression of translation when short double-stranded RNA molecules, a few hundred nucleotides in length and containing the same sequence as a target mRNA, were introduced into cells. The effect was dramatic: most of the mRNA with the target sequence was quickly destroyed. The current mechanistic model of RNAi repression is that first, the double-stranded molecules are cleaved by an endonuclease called Dicer, which cleaves with over-hanging single-stranded 3’ ends. This allows the short fragments (siRNA, ~20nt long) to form a complex with several proteins (RISC, RNA-induced silencing complex). The RISC splits the double-stranded fragments into single strands, one of which is an exact complement to the mRNA. Because of the complementarity, this is a stable interaction, and the double-stranded region appears to signal an endonuclease to destroy the mRNA/siRNA hybrid.
The final method of controlling levels of gene expression is control after the fact, i.e., by targeted destruction of the gene product protein. While some proteins keep working until they fall apart, others are only meant for short-term use (e.g. to signal a short phase in the cell cycle) and need to be removed for the cell to function properly. Removal, in this sense, would be a euphemism for chopped up and recycled. The ubiquitin-proteasome system is a tag-and-destroy mechanism in which proteins that have outlived their usefulness are polyubiquitinated. Ubiquitin is a small (76 amino acids, ~5.6 kDa), highly conserved (96% between human and yeast sequences) eukaryotic protein (fig. 9) that can be attached to other proteins through the action of three sequential enzymatic steps, each catalyzed by a different enzyme.
Figure 9. Ubiquitin. This 3D representation was generated from the file 1ubi (synthetic human ubiquitin) in the RCSB Protein Data Bank
E1 activates the ubiquitin by combining it with ATP to make ubiquitin-adenylate, and then transfers the ubiquitin to itself via a cysteine thioester bond. Through a trans(thio) esterification reaction, the ubiquitin is then transferred to a cysteine in the E2 enzyme, also known as ubiquitin-conjugating enzyme. Finally, E3, or ubiquitin ligase, interacts with both E2-ubiquitin and the protein designated for destruction, transferring the ubiquitin to the target protein. After several rounds, the polyubiquitinated protein is send to the proteasome for destruction.
Mutations in E3 genes can cause a variety of human medical disorders such as the neurodevelopmental disorders Angelman syndrome, Hippel-Lindau syndrome, or the general growth disorder known as 3-M syndrome. Mechanisms linking malfunction in ubiquitination pathways and symptoms of these disorders are not currently known.
Figure 10. Polyubiquitination of a targeted protein (blue) requires three ubiquitinating enzymes, E1, E2, and E3. Once tagged, the protein is positioned in the proteasome by binding of the polyubiquitin tail to the outer surface of the proteasome. The proteasome then cleaves the protein into small polypeptides.
Proteasomes are very large protein complexes arranged as a four-layered barrel (the 20S subunit) capped by a regulatory subunit (19S) on each end. The two outer rings are each composed of 7 α subunits that function as entry gates to the central rings, each of which is composed of 7 β subunits, and which contain along the interior surface, 6 proteolytic sites. The 19S regulatory units control the opening and closing of the gates into the 20S catalytic barrel. The entire proteasome is sometimes referred to as a 26S particle.
A polyubiquitinated protein is first bound to the 19S regulatory unit in an ATP-dependent reaction (the 19S contains ATPase activity). 19S unit opens the gates of the 20S unit, possibly involving ATP hydrolysis, and guides the protein into the central proteolytic chamber. The protease activity of proteasomes is unique in that it is a threonine protease, and it cuts most proteins into regular 8-9 residue polypeptides, although this can vary.
As we will see in the cell cycle chapter, proteasomes are a crucial component to precise regulation of protein functions.