Regulation of Translation

Regulation of Translation Global Regulation of Translation Initiation in Bacteria and Eukaryotes Translation is globally regulated in response to amino acid starvation Protein synthesis in the cell needs to be globally regulated in response to external conditions and stimuli. Protein synthesis rates should be high when nutrients are plentiful to allow maximal cell growth, and low in times of nutrient deprivation Under normal circumstances, transfer RNAs (tRNAs) are immediately aminoacylated once released from a round of elongation on the ribosome and are bound in the cell by EFTu (or eEF1A) once they are loaded with the appropriate amino acid As such, uncharged tRNA - a tRNA without an amino acid attached - is one indicator of low levels of amino acid and a trigger for global shutdown of protein synthesis in bacterial and eukaryotic cells In bacteria, the uncharged tRNA induces a stress response when it begins to compete with decreasing concentrations of ternary complex (EFT-guanosine triphosphate (GTP)-aa-tRNA) and binds directly to the ribosomal A site More importantly, however, this binding event on the ribosome recruits a protein called RelA to the complex. Once recruited, RelA synthesizes high concentrations of (p)ppGpp (a pentaphosphate guanine nucleotide alarmone known as "magic spot") from GTP/GDP and ATP precursors The nucleotide (p)ppGpp itself has profound effects on transcription, which include a decrease in the overall synthesis of stable RNAs (such as ribosomal RNA (rRNA) and tRNA) and an increase in the synthesis of stress response factors that go about the task of replenishing the cell's declining resources There [in eukaryotes], the uncharged tRNA binds to a protein Gcn2 (in yeast), and more specifically to a domain in this protein related to the histidyl-tRNA synthetase Phosphorylation of eIF2 increases its affinity for its GEF, eIF2B, to the point where active eIF2 is effectively depleted from the cell and overall initiation is slowed However, as we saw in bacteria, the cells also respond to this stress by inducing the expression of certain genes that are important for restoring cellular resources Much global translational control is achieved through modulation of eIF2 and its GEF partner eIF2B Phosphorylation of eIF2, is a common means of affecting the equilibrium between free eIF2 and eIF2B:eIF2; the specific phosphorylation of serine 51 on the alpha subunit of eIF2 results in the constitutive binding of eIF2-GDP to eIF2B This binding interaction sequesters the smaller amount of eIF2B present in the cell relative to eIF2 and prevents it from catalyzing nucleotide exchange In the absence of eIF2B activity, eIF2 remains in a GDP-bound state and unable to participate in further rounds of initiation eIF2B comprises five subunits, three of which bind eIF2 and mediate tight binding of the Ser51 phosphorylated form, whereas the other two subunits conduct the GDP-GTP exchange reaction The mRNA-bound cap complex initiation factors are also commonly targeted to modulate global translation in eukaryotes Generally, mRNAs are at a higher concentration than the components of the translational machinery, and therefore mRNAs most compete for the available initiation factors if they are to be translated First, the transcription of eIF4E is regulated in response to external stimuli. Second, eIF4E itself is phosphorylated in response to external stimuli, which is thought to decrease the affinity for the 5’ cap of mRNA, and thus lead to globally decreased initiation Finally, eIF4E can bind proteins known as 4E-BPs (eIF4E-binding proteins) which compete with eIF4G for binding to eIF4E and thereby impede assembly of the closed loop formation at the cap Regulation of translation initiation by cis-acting sequences in the 5' untranslated region in bacteria and eukaryotes Gene-specific translational regulation depends on sequence features in the mRNA The rate of synthesis of a protein depends on many factors: the rate of transcription of the gene, the stability of the mRNA, and the rate of its translation, to name the most obvious Arguably, the most rapid changes in the rates of protein synthesis can be achieved by regulating the rate of translation of an mRNA population that is already present in the cell; in this case, there is no requirement to produce new mRNA to increase the downstream step of protein synthesis In bacteria, the best known examples of the regulation of initiation involve direct obstruction of the Shine-Dalgarno sequence in the 5' untranslated region (UTR) of an mRNA, preventing the formation of competent initiation complex Shine-Dalgarno sequestration is a common mechanism for the regulation of translation in bacteria The expression of PrfA, a key transcriptional activator of genes required for infection by this organism, is regulated by temperature-dependent changes of the structure of the 5' UTR At low temperatures outside host animals, the Shine-Dalgarno sequence of the prfA mRNA is locked in an RNA structure that renders it inaccessible to the ribosome, thus blocking translation Once a bacterium enters a host, however, temperature-dependent structural reorganization of this region of the mRNA makes the Shine-Dalgarno sequence accessible to the ribosome, and translation of the prfA mRNA can occur There are a growing number of examples where the expression of biosynthetic genes is subject to autoregulation - that is, where the end-product of the pathway directly regulates its own synthesis In bacteria, the small molecule product often binds to the mRNA sequence upstream of the open reading frame (ORF) and changes the structure of the mRNA. Such elements are generally referred to as riboswitches It is plausible (and even likely) that metabolite-sensing systems, based simply on structural changes in RNA, are remnants of ancient regulatory systems that evolved in the RNA world, before the evolution of regulatory proteins A class of small regulatory RNAs (generally 100 - 150 nucleotides in length) in bacteria also promotes or limits translation by directly base-pairing with the 5' UTR, such that the Shine-Dalgarno sequence is either sequestered or revealed Initiation in eukaryotes can be regulated by blocking access to the ribosome-binding site Inside cells, iron is bound to a storage protein known as ferritin; the higher the concentration of iron in a cell, the more ferritin the cell requires When iron is scarce in the cell, the IRPs (iron regulatory proteins) bind to the IRE (iron response element). This binding prevents the initiation of translation by preventing ribosome access to the AUG start site, probably by blocking the scanning ribosome Upstream ORFs regulate the synthesis of Gcn4 protein in yeast Under all conditions - whether the cell is starved for amino acids or not - uORF1 is effectively translated. What happens beyond uORF1, however, differs, depending on the cellular levels of eIF2 available Central to all possible outcomes is that the uORF1 stop codon (and the sequence context in which it is located) favors the resumption of scanning following termination, rather than full ribosome drop-off and recycling - that is, the 40S subunit remains associated with the mRNA and travels further along the mRNA where it can encounter downstream sequence elements Unlike uORF1, however, the sequence context of the stop codons for these uORFs (2-4) does not favor the reinitiation of scanning and complete ribosome recycling (that is, including dissociation of the 40S subunit) happens more efficiently Incidentally, the GCN4 gene start site is thought to be a relatively robust one, thus ensuring that ribosomes do not easily scan past it as well. Consequently, Gcn4 protein is translated at high levels Regulation of Translation Initiation Through Cis-Acting Sequences in the Three prime UTR in Eukaryotes Polyadenylation levels can be regulated by 3' UTR binding proteins The Xenopus oocyte contains maternally derived mRNAs that are not immediately translated into protein - these mRNAs are said to be dormant or translationally repressed Dormant mRNAs have short poly(A) tails, which only during maturation are extended to allow the mRNAs to be translated into protein The 3' UTRs of the dormant mRNAs have sequences referred to as cytoplasmic polyadenylation elements (CPEs), which play a key role in controlling their expression The interaction of eIF4E with Maskin directly precludes the binding of eIF4G to eIF4E, thus "masking" the expression of an mRNA by preventing formation of the closed circle initiation complex Maskin is a type of 4E-BP with additional binding specificity for the protein CPEB that allows for genespecific regulation through 3' UTR recruitment This rather complicated sequence of events allows mRNAs that are held dormant by the CPE sequence element to be activated by a single event - the activation of the kinase Eg2 RNAs or proteins bound to the 3’ UTR can regulate translation at multiple steps following polyadenylation The expression of many genes in eukaryotic systems can be controlled by other 3' UTR regulatory sequences, and often the regulation is applied to different stages in the in Manson (or even elongation) process Finally, the microRNA (miRNA) class of small RNAs in eukaryotes is proposed to regulate the translation of specific mRNAs. These small RNAs are known to act through binding interactions with partially complementary sequences in the 3' UTRs of genes Regulation of Translation Elongation in Bacteria and Eukaryotes Ribosomes can be stalled by phosphorylation of eEF2 in response to stress In one well-characterized pathway in yeast, hyper-osmotic stress triggers the so-called Hog1 kinase (a homolog of the MAP kinase p38 in higher eukaryotes), which phosphorylates Rck2 (similar to CaM kinase in higher eukaryotes), which, in turn, phosphorylates eEF2 The result of this modification is that pre-translocation-state ribosomes accumulate in the cell and elongation is thus inhibited Elongation regulation commonly accompanies initiation regulation, as cells attempt to comprehensively stop the energetically consumptive process of translation in order to redirect their limited resources Translation elongation of specific mRNAs can be stalled by nascent peptidemediated interactions Both in bacteria and eukaryotes, there are several examples of peptide-mediated stalling where certain sequences in ORFs lead to stalling, regulating expression of the specific mRNAs that encode them The coupling of transcription and translation is a common theme in bacteria that lack a nucleus that spatially divides these two processes