Gene Control Mechanisms at Transcription and Translation Levels Dr. M. Vijayalakshmi School of Chemical and Biotechnology SASTRA University Joint Initiative of IITs and IISc Funded by MHRD Page 1 of 9
Table of Contents 1 INTRODUCTION... 3 1.1 TRANSCRIPTION CONTROL... 4 1.2 POST TRANSCRIPTIONAL CONTROL... 6 1.3 TRANSLATIONAL CONTROL... 6 1.4 POST TRANSLATIONAL CONTROL... 7 2 REFERENCES... 9 2.1 TEXT BOOKS... 9 2.2 LITERATURE REFERENCES... 9 Joint Initiative of IITs and IISc Funded by MHRD Page 2 of 9
1 Introduction Fig 1. Mechanisms of gene regulation in eukaryotes The complicated mechanisms of mrna formation, the complex structures of the nucleus and cytoplasm of eukaryotic cells, the robust genetic program involved in differentiation of eukaryotic cells strikingly differentiate eukaryotic gene control from bacterial gene regulation. As stated earlier, the eukaryotic genes undergo several more steps of regulation than the bacterial genes. Single molecule experiments have clearly and accurately decipher the mechanical steps involved in eukaryotic gene control. Such experiments have even determined at the transcription level, the amount of specific mrna molecules that are regulated, the processing of the nuclear RNA and the stability of mrna. As stated in the previous class, gene regulation can be brought about by altering gene expression states in a cell. This can be effected at the levels of transcription and translation and during post transcription and post translational events. Joint Initiative of IITs and IISc Funded by MHRD Page 3 of 9
1.1 Transcription Control Transcription in eukaryotic cells requires at the first level of control the choice of RNA polymerases. Eukaryotes have three different RNA polymerases, polymerases I, II and III. In vivo and in vitro experiments have shown that the RNA polymerase II initiates transcription from a site located near a conserved 8 to 10 nucleotide region, 25 to 30 nucleotides upstream (5 ) from the RNA start site. The nucleotides TATA which are strongly conserved at this site gives the name TATA box or Goldberg-Hogness box. There are other sequences equally important in regulating the access polymerases to the DNA. Transcription factors play a key role in helping RNA polymerase to bind to promoter region. Transcription factors bind to various consensus sequences like TATA box in Fig 2. TATA binding protein recognises this box and recruits RNA polymerase II to promoter site for transcription initiation. Transcription factors called activators bind to enhancers. Enhancers lie far from the transcription start site and are position insensitive, while the promoter sites lie close to transcription start site and are position sensitive. Genes lacking TATA box have initiator sequence for initiating transcription. Combination of specific transcription factors is critical for the transcription of a particular gene. Insulators are DNA sequences regulating gene expression. They play a key role in enhancer blocking. Positional enhancer blocking occurs only when insulator lies in between enhancer and operator. Insulators allow genes to remain independent in the genome by restraining the unnecessary signals influencing the gene. Joint Initiative of IITs and IISc Funded by MHRD Page 4 of 9
Fig 2: Factors that influence the regulation of a typical gene X A variety of structural motifs present in transcription factors interact with DNA at specific sequences. On the basis of DNA binding domains they accommodate, eukaryotic transcription factors are classified as Zinc Finger proteins which have regions folding around a central Zn 2+ ion. Generally DNA binding domains of transcription factors contain such regions. Proteins which do not bind to DNA also show Zn finger patterns. Leucine Zipper proteins have a leucine at every seventh position on the protein sequence. Such proteins function as transcription factors when present in the form of a dimer. The presence of Leucine helps in parallel zippering of dimer. Yeast Gcn4 is an example of such protein. Helix turn helix motif is composed of two alpha helices connected by a short amino acid strand. One of them is an amino terminal helix and the other a C- terminal helix. The C-terminal helix is critical for DNA recognition while the other helix stabilizes the DNA- protein interaction. The C-terminal helix binds DNA at major grooves through hydrogen bonding. The Cro, lambda repressor and Catabolite gene activator protein are examples of Helix turn helix motif structures. Joint Initiative of IITs and IISc Funded by MHRD Page 5 of 9
Hormones play a crucial role in gene transcription. Type 1 hormones like gluco corticoids being hydrophobic bind to intracellular receptors located in cytoplasm. Hormone receptor interaction causes dissociation of the nuclear localisation signal of receptor which is then transported to nucleus where it binds to hormone response elements to cause transcription of gene. Type II hormones like epinephrine being hydrophilic bind to cell surface receptors and activate G- proteins, leading to gene transcription. 1.2 Post Transcriptional control Post transcriptional control comes into play after the primary transcript is produced. Alternative mrna splicing is one of the post transcriptional regulatory methods and follows tissue specific or developmental fashion. Tissue specific regulation results in the formation of two different proteins from the same gene in different cells while differentiation specific regulation results in the formation of more than one functionally different protein in the same cell. The best example is the production of calcitonin in thyroid cells, but calcitonin related peptide in brain cells due to alternative splicing. Alternative splicing results in different combinations of exons producing different proteins from the same transcript. Alternative splicing has a switching function which leads to functional or inactive proteins. 1.3 Translational control This mechanism provides hindrance to protein synthesis through proteins involved in translation. This includes stability of mrna, probability of translation initiation apart from regulation of overall protein synthesis. These mechanisms control the amount of protein produced from mrna during a translational event. Stability of mrna is important step that controls protein synthesis. Usually the eukaryotic mrna survives for 3 hours but in certain cases where the amount of protein required in higher amounts, the mrna survives for several days. In silk worms which produce fibroin, the amount of fibroin for cocoon formation takes days thus the fibroin mrna survives for several days.repeated translation of Joint Initiative of IITs and IISc Funded by MHRD Page 6 of 9
mrna results in the formation of 10 5 fibroin molecules from 10 4 fibroin mrnas. Oviduct cells synthesize ovalbumin in chickens but such cells have single copy of this gene per haploid dose. Thus in such circumstances the lifetime of mrna should be high so that same mrna is translated repeatedly to achieve the desired protein level. Reticulocytes synthesize a single type of protein. In such circumstance translation is regulated by modulating total protein synthesis. Globin synthesis in rabbit reticulosites is controlled at the level of translation. Such cells lack nucleus and thus cannot be controlled at the transcriptional level. The process of translation requires elongation factor eif2 and eif2 stimulating protein (ESP) besides other molecular players that regulate the event. 1.4 Post Translational control This control operates after the protein is synthesised in the cell. Post translational modifications include covalent modifications which occur in proteins after translation. Such modifications add another layer of complexity to the complexity embedded in the genome. Post translational modifications play a critical role in regulating activity, localization, and enabling inter molecular interactions inside the cell. Glysosylation regulates protein stability, influences protein folding kinetics. Ubiquitylation of proteins acts as a signal for proteasomal degradation of proteins. Ubiquitin is a composed of 76 amino acid residues and links to proteins via isopeptide bond. S-nitrosylation is another vital posttranslational modification which modulates protein stability. Caspases residing in the outer mitochondrial membrane are present as S-nitrothiols. On apoptotic signalling, they translocate to the cytosol, become denitrosylated and are activated in the cytosol. Post translational modifications like methylation, acetylation are also involved in diseased states. We thus understand that, gene expression can be controlled intricately by targeting either the transcription machinery or the translation machinery in a Joint Initiative of IITs and IISc Funded by MHRD Page 7 of 9
spatio temporal manner. Controls exercised at post transcription and post translation levels have vital implications during disease signalling. This takes us to an interesting logical question on whether apart from the controls exercise at theses states can be totally shut off or activate a gene to initiate favorable responses inside the cell. We shall discuss these in detail by initiating the next lecture on genetic switches, whose systems level understanding has been extensively studied. Joint Initiative of IITs and IISc Funded by MHRD Page 8 of 9
2 References 2.1 Text Books 1. David S. Latchman, Gene Control, 1/e, Garland Science Publications, (2010). 2. Alberts B. Jhoson A, Lewis J, Molecular Biology of the cell, 4/e, (2002). 2.2 Literature References 1. D A Day and M F Tuite, Post-transcriptional gene regulatory mechanisms in eukaryotes: an overview, Journal of Endocrinology (1998), 157, 361-371. 2. Daniel S. Oppenheim and Charles Yanofsky, Translational Coupling During Expression Of The Tryptophan Operon Of Escherichia Coli, Genetics, (1980), 95, 785-795 3. Daniel H. Lackner et al., Translational Control of Gene Expression: From Transcripts to Transcriptomes, International Review of Cell and Molecular Biology, (2008), 271, 200-238. Joint Initiative of IITs and IISc Funded by MHRD Page 9 of 9