Mammalian ribosomal protein (rp) mRNAs are subject to translational control, as illustrated by their selective release from polyribosomes in growth-arrested cells and their underrepresentation in polysomes in normally growing cells. In the present experiments, we have emined whether the translational control of rp mRNAs is attributable to the distinctive features of their 5' untranslated region, in particular to the oligopyrimidine tract adjacent to the cap structure. Murine lymphosarcoma cells were transfected with chimeric genes consisting of selected regions of rp mRNA fused to non-rp mRNA segments, and the translational efficiency of the resulting chimeric mRNAs was assessed in cells that either were growing normally or were growth-arrested by glucocorticoid treatment. We observed that translational control of rpL32 mRNA was abolished when its 5' untranslated region was replaced by that of .8-actin. At the same time, human growth hormone (hGH) mRNA acquired the typical behavior of rp mRNAs when it was preceded by the first 61 nucleotides of rpL30 mRNA or the first 29 nucleotides of rpS16 mRNA. Moreover, the translational control of rpSl6-hGH mRNA was abolished by the substitution of purines into the pyrimidine tract or by shortening it from eight to six residues with a concomitant cytidine -* uridine change at the 5' terminus.These results indicate that the 5'-terminal pyrimidine tract plays a critical role in the translational control mechanism. Possible factors that might interact with this translational cis regulatory element are discussed.Control at the translational level plays a dominant role in the regulated expression of eukaryotic ribosomal protein (rp) genes under a variety of conditions (1-3). These include: early development of Dictyostelium discoideum (4), Drosophila melanogaster (5)
The promoters of several eukaryotic genes transcribed by RNA polymerase H contain elements located downstream of the transcriptional start site. To gain insight into how these elements function in the formation of an active transcription complex, we have cloned and sequenced the cDNA that encodes 8, a protein that binds to critical downstream promoter elements in the mouse ribosomal protein rpL30 and rpL32 genes. Our results revealed that the 6 protein contains four C-terminal zinc fingers, which are essential for its DNA binding capability and a very unusual N-terminal domain that includes stretches of 11 consecutive negatively charged amino acids and 12 consecutive histidines. The sequence of the 8 protein was found to be essentially identical to a concurrently cloned human transcription factor that acts both positively and negatively in the context of immunoglobulin enhancers and a viral promoter. Our structural modeling of this protein indicates properties that could endow it with exquisite functional versatility.that encodes a protein termed 6, which binds to critical downstream elements in the genes encoding mouse ribosomal proteins L30 and L32 (rpL30 and rpL32) (Fig. 1A) and to downstream elements of several other genes, as inferred from sequence comparisons of functionally important factor binding sites (Fig. 1B) and a direct competition assay (10).tAt the completion of this study, we learned that the 6 sequence is almost identical to that of a human transcription factor that can act either negatively or positively in the context of the immunoglobulin K 3' and heavy-chain enhancers (13) and the P5 promoter of adeno-associated virus (14). The properties of this protein apparently suit it to function with downstream promoter elements, as well as to perform both activating and repressive functions. Our results have revealed that 8 is a zinc finger protein with unusual structural features that could endow it with exquisite functional versatility.The transcription of eukaryotic genes by RNA polymerase II is regulated by an interplay of sequence-specific activator or repressor factors and essentially nonspecific general factors and chromatin components (1). The specific factors recognize sequence modules or elements that are situated both in promoters-i.e., regions near the transcriptional start siteand in enhancers-i.e., regions distant from the start site. Specificity in transcriptional regulation is determined by the composition and organization of these elements and the availability of the factors that interact with them.A considerable effort is currently being made to understand the way in which sequence-specific and general factors interact during formation of active transcription complexes (2, 3). In most mechanistic studies of promoter function, major attention has been given to factors that bind to elements located upstream of the transcriptional start site. However, it has become increasingly evident that a large number of both cellular and viral genes utilize elements that are located downst...
The importance of glucokinase (GK; EC 2.7.1.12) in glucose homeostasis has been demonstrated by the association of GK mutations with diabetes mellitus in humans and by alterations in glucose metabolism in transgenic and gene knockout mice. Liver GK activity in humans and rodents is allosterically inhibited by GK regulatory protein (GKRP). To further understand the role of GKRP in GK regulation, the mouse GKRP gene was inactivated. With the knockout of the GKRP gene, there was a parallel loss of GK protein and activity in mutant mouse liver. The loss was primarily because of posttranscriptional regulation of GK, indicating a positive regulatory role for GKRP in maintaining GK levels and activity. As in rat hepatocytes, both GK and GKRP were localized in the nuclei of mouse hepatocytes cultured in low-glucose-containing medium. In the presence of fructose or high concentrations of glucose, conditions known to relieve GK inhibition by GKRP in vitro, only GK was translocated into the cytoplasm. In the GKRP-mutant hepatocytes, GK was not found in the nucleus under any tested conditions. We propose that GKRP functions as an anchor to sequester and inhibit GK in the hepatocyte nucleus, where it is protected from degradation. This ensures that glucose phosphorylation is minimal when the liver is in the fasting, glucose-producing phase. This also enables the hepatocytes to rapidly mobilize GK into the cytoplasm to phosphorylate and store or metabolize glucose after the ingestion of dietary glucose. In GKRP-mutant mice, the disruption of this regulation and the subsequent decrease in GK activity leads to altered glucose metabolism and impaired glycemic control. G lucokinase (GK; EC 2.7.1.12), the principal hexokinase in liver parenchymal cells and in pancreatic  cells, is a critical component of the physiological glucose-sensing apparatus (1). In humans, GK heterozygous mutations lead to the autosomal, dominant, maturity-onset diabetes of the young (MODY) (2, 3) phenotype. In mice, changes in GK activity by gene knockout and by overexpression resulted in altered glucose homeostasis and demonstrated that modest changes in liver GK activity alone were sufficient to cause significant alterations in blood glucose levels (4-9). Liver GK activity has been reported to be lower in some obese humans with type 2 diabetes mellitus when compared with nondiabetic normal weight or obese individuals, suggesting a role for liver GK in glucose homeostasis in type 2 diabetes (10).In the liver, GK activity is allosterically inhibited by GK regulatory protein (GKRP) (11-13). This protein, found in the livers of all animal species where GK is present, shows no inhibitory effect on other known hexokinases (11-13). In rodents, GKRP inhibition of GK is relieved by high concentrations of glucose and by fructose 1-phosphate, and is potentiated by fructose 6-phosphate (11-13). Recent studies have demonstrated that both GK and GKRP are in the nucleus of rat hepatocytes cultured in a medium containing 5.5 mM glucose (14,15). When the growth medium w...
BackgroundSouth Asians are more insulin resistant than Europeans, which cannot be fully explained by differences in adiposity. We investigated whether differences in oxidative capacity and capacity for fatty acid utilisation in South Asians might contribute, using a range of whole-body and skeletal muscle measures.Methodology/Principal FindingsTwenty men of South Asian ethnic origin and 20 age and BMI-matched men of white European descent underwent exercise and metabolic testing and provided a muscle biopsy to determine expression of oxidative and lipid metabolism genes and of insulin signalling proteins. In analyses adjusted for age, BMI, fat mass and physical activity, South Asians, compared to Europeans, exhibited; reduced insulin sensitivity by 26% (p = 0.010); lower VO2max (40.6±6.6 vs 52.4±5.7 ml.kg−1.min−1, p = 0.001); and reduced fat oxidation during submaximal exercise at the same relative (3.77±2.02 vs 6.55±2.60 mg.kg−1.min−1 at 55% VO2max, p = 0.013), and absolute (3.46±2.20 vs 6.00±1.93 mg.kg−1.min−1 at 25 ml O2.kg−1.min−1, p = 0.021), exercise intensities. South Asians exhibited significantly higher skeletal muscle gene expression of CPT1A and FASN and significantly lower skeletal muscle protein expression of PI3K and PKB Ser473 phosphorylation. Fat oxidation during submaximal exercise and VO2max both correlated significantly with insulin sensitivity index and PKB Ser473 phosphorylation, with VO2max or fat oxidation during exercise explaining 10–13% of the variance in insulin sensitivity index, independent of age, body composition and physical activity.Conclusions/SignificanceThese data indicate that reduced oxidative capacity and capacity for fatty acid utilisation at the whole body level are key features of the insulin resistant phenotype observed in South Asians, but that this is not the consequence of reduced skeletal muscle expression of oxidative and lipid metabolism genes.
The promoters of the mouse ribosomal protein genes rpL30, rpL32, and rpS16 are of equal strength, as indicated by in vivo measurements of polymerase loading and by their relative efficiency in driving the expression of a linked reporter gene. The equipotency of these promoters appears to derive from a remarkably similar architecture in which five or more elements are distributed over a 200-bp region that spans a polypyrimidineembedded cap site. Three trans-acting factors are shared by the rpL30 and rpL32 promoters, one of which, 8, recognizes a common CNGCCATCT motif in the first (untranslated) exons. Site-specific mutagenesis demonstrated that 8-factor binding is critical for rpL30 promoter function. The repeated occurrence of this novel promoter architecture among ribosomal protein genes with very different coding specificities is most readily explained by convergent evolution.
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