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Gene regulation in eukaryotes Table of content 0. Introduction 0-1. The importance of genetic regulation 0-2. Regulatory levels I. Regulation of transcription I-1. Chromatin regulation I-2. Interactions between cis- and trans-regulatory elements I-2.1. Structure of a gene I-2.2. Promoters I-2.3. Enhancers and silencers I-2.4. The pre-initiation complex I-2.5. Transcription factors I-2.6. Types of gene expression I-2.7. cell communication-induced gene expression I-2.8. Steroid hormone activation I-2.9. Interferon- activation I-2.10. Cell type-specific gene expression I-2.11. Interactions among gene expressions I-2.12. Evolution of genetic regulation II. Regulation after transcription II-1. Posttranscriptional regulation II-1.1. mRNA processing II-1.2. Splicing II-1.3. Capping II-1.4. Polyadenylation II-1.4. mRNA editing II.1.5. Alternative gene usage II-1.6. mRNA transport II-1.7. mRNA localization II-1.8. mRNA stability II-2. Regulation of translation II-3. Posttranslational regulation II-3.1. Protein degradation II-3.2. Protein processing and modification III. Regulatory RNAs III-1. Micro RNAs III-2. Overlapping RNAs III-3. RNA interference 1 0. Introduction (0. + 1. slides) 0-1. The importance of genetic regulation Gene expression has become a key issue in biology in the past couple of years. There are several reasons for this. Our curiosity to understand the processes (1) controlling the formation of an adult human from a single cell; (2) controlling the operation of adult body; (3) and resulting in the amazing variability observable in human population and in other animals. Understanding genetic regulation is basically important in future medicine: in diagnosis and cure of illness, and in general, in individual-based healthcare. The technological development has enabled us by now to accurately analyze both the expression of individual genes and simultaneously a huge number of genes (functional genomics*: DNA chips* and protein chips*). The other reason for the present day popularity of gene expression is based on two novel scientific discoveries. One of the discoveries includes the (1) regulatory RNAs* (noncoding RNAs). According to the traditional view, the exclusive function of RNAs is to convey information from DNA to proteins. However, it turned out that RNAs operate not only as mediators in the flow of genetic information but they also regulate the manifestation of this information. To be more specific, it appears that a great portion of genes are under the control of various types of regulatory RNAs. (2) It also turned out that the various anatomical structures and physiological processes are determined by specific genetic modules, termed gene networks* composed of functionally-linked genes, and not by individual genes. The importance of genetic regulation is indicated that in 2006 both in Chemistry and Physiology or Medicine the Nobel Price was awarded in this issue. 0-2. Regulatory levels (1. + 2. slides) Transcription mRNA processing mRNA transport mRNA localization mRNA stability post-transcriptional regulation Translation Protein degradation Protein processing and modification post-translational regulation 2 I. Regulation of transcription 1. Chromatin* regulation (3. slide) - DNA methylation - Acetylation - Histone-repulsing sequences 2. Interactions between cis- and trans- regulatory elements ● cis-elements: promoters, enhancers and silencers ● trans-elements: RNA polymerase, transcription factors and co-factors I-1. Regulation of chromatin (4. slide) Regulation of histone – DNA binding allows the establishment of different chromatin states leading to distinct ‘readouts’ of the genetic information, such as gene activation or gene silencing. The acetylation of histone proteins removes positive charges, thereby reducing the affinity between histones and DNA, which makes it easier for RNA polymerase and transcription factors to find access to regulatory sequences and to activate transcription from them. Methylation of histones and DNA sequences leads to the opposite effect than that of acetylation. Methylation of histones results in a stronger binding to DNA. Methylation of DNA sequences has been shown to result in the silencing of gene expression in certain cells. Some genes and gene clusters are bracketed by so called nucleosomes repulsing sequences, such as matrix attachment region (MAR), locus control region (LCR) and scaffold-associated region (SAR) sequences. These sequences have been shown to inhibit chromatin condensation via their repulsive effects on the nucleosomes, thereby allowing transcription from the DNA stretches they bracket I-2. Interactions between cis- and trans- regulatory elements Cis-element: they are found on he same DNA strand as the gene Trans-element: they are located in the cytoplasm I-2.1. structure of a gene (5. slide) A gene is composed of transcribed and regulatory regions. The transcribed region consists of non-coding (5’-UTR and 3’-UTR; untranslated regions) and coding region. In a strict sense, only the exons (encoding amino acid sequences) are called as coding regions. The parts of regulatory regions are the promoters, enhancers (facilitates transcription) and silencers (inhibit transcription). Introns are 11 times longer than exons on average, while non-coding exons (5’UTR and 3’-UTR) have the same size as the coding ones. I-2.2. Promoters (6. slide) Transcriptional regulation is by far the most important mode for the control of eukaryotic gene expression. The cis-regulatory sequences involve regulatory DNA motives, which are recognized by specific transcription factors. Basal promoter elements termed TATA boxes are located between 20 and 30 bases upstream of the transcriptional start site of eukaryotic genes. Proximal promoter elements, such as the CAAT box and GC box, reside within 40 to 250 bases upstream of the transcriptional start site. The various “boxes” are so called consensus sequences meaning that that they do not or only slightly vary even across large evolutionary distances. 3 I-2.3. Enhancers and silencers (7. slide) The enhancer/silencer regulatory sequences are predominantly located upstream of the genes, though some elements may occur downstream or within the introns. Enhancer sequences can reside up to hundreds of thousands of base pairs from the coding region. The regulatory sequences of an average gene reside within 10.000 base pairs. The number and type of regulatory elements vary with each gene. The borders of genes are determined by the insulator sequences, whose function is to restrict the effect of regulatory sequences to the gene they control and isolate. I-2.4. The pre-initiation complex (8. slide) The pre-initiation complex facilitates the binding of RNA polymerase II to the promoter, and thus the transcription. The RNA polymerase is composed of 12 subunits. RNA polymerase binding the promoter has to be preceded by the attachment of several transcription factors to the promoter or to the polymerase itself. The pre-initiation complex can only initiate a basal expression level from a specific gene. I-2.5. Transcription factors (9. slide) Trans-regulatory factors of transcription, which exert direct regulatory effects on the gene expression, include RNA polymerases, transcription factors and accessory factors. They can directly bind to the DNA or to another transcription factor. Approximately 5-10 percent of total gene content encodes transcription factors in higher-order organism, including human. The different classes of eukaryotic RNAs are transcribed by three distinct polymerases. The most complex controls are those that regulate the expressions of RNA polymerase IItranscribed mRNAs. Numerous proteins, such as TFIIA, B, C, D (TF = transcription factor) interact with the TATA-box in a direct or indirect manner. The protein identified as C/EBP binds to the CAAT-box element, and the SP1 protein binds to the GC-box. Several types of transcription factors are responsible for the recognition of more distant enhancer elements. The various cell types each express characteristic combinations of transcription factors, and this comprising the major mechanism ensuring tissue-specific gene expression. Transcription factors bind DNA either as homodimers or as heterodimers, with a variety of partners, with distinct consequences for transcription. Further, the activities of transcription factors depend on the presence or absence of cofactors, and on post-translational modifications, including phosphorylation, acetylation and glycosylation. An additional means of transcription regulation is the alternative usage of promoters, which enforces the alternative use of exons and can result in a variety of tissue-specific isoforms. I-2.6. Types of gene expression (10. slide) 1. Constitutive (continuous) – e.g. housekeeping genes 2. Induced 2a. nutrition material-induced: glucose in liver cell, (E.coli: lac operon) 2b. stress: heat shock, osmotic shock (salt) 2c. Cell-communication-induced: hormones, growths factors 2d. Developmentally regulated I-2.7. Cell communication-induced gene expression (11. slide) Cells more often communicate with each other by means of signal molecules. A signal molecule can bind to a receptor inducing a strictly regulated cascade of biochemical events, called signal transduction. Alternatively, signal molecules can enter the cell and exert their effects in the cytoplasm or in the nucleus. There are three basic types of them. They can be 4 transcription factors, thus they directly influence gene expression by binding an enhancer sequence on he DNA (it is rare), or they can bind to a transcription factor, or to an other factor, which exert its effect on a transcription factor through multiple steps. The intracellular binding partner of a signal molecule is also called receptor. I-2.8. Steroid hormone activation (12. slide) The glucocorticoid receptors are located in the cytoplasm in an inactive state (hsp29 chaperon performs inhibition). Steroid hormone binding dislocates hsp29, and results in the formation of a dimeric (two-subunit) molecule, which in turn, enter to the nucleus, bind to its response DNA element (GRE: glucocorticoid response element) and activate transcription from the linked gene. I-2.9. Interferon- activation (13. slide) Interferon (IFN)-γ binding to its receptor induces JAK kinase activation, which in turn results in the phosphorylation of STAT-1 transcription factor. As a result, activated STAT-1 forms a dimeric molecule, enter to nucleus and induce transcription from genes harboring the appropriate response elements (alternative terms: recognition sequences, motives, consensus sequences). I-2.10. Cell type-specific gene expression (14. slide) Although, almost all of our cells comprise the same genetic content, there are a huge number of cell type, and each type of cells expresses different genes. The question is how it is possible. The various cell types developed by means of differentiation. The genetic basis of differentiation is the formation of different chromatin pattern (varying histone binding to the DNA) in different tissues. The histone binding pattern determines the type of transcription factors expressed in a cell, and the transcription factors decide whether the expression of a particular gene is On or OFF. Normally, several transcription factors and co-factors are required for the control of a gene. If one of them is missing, there is no transcription. Further, even though, the appropriate transcription factors are present in a cell, no gene expression is induced if the regulatory region of the target gene is blocked by histones. I-2.11. Interactions among gene expressions (15. slide) A particular physiological process is controlled by functionally linked groups of genes, which is called gene network. These gene directly or indirectly interact with each other. There can be various results of the interactions. An extreme situation is when the changing of expression of s ingle gene alters the expression of all members of the gene cluster. Naturally, it is not a realistic situation, especially in embryogenesis, since the expression of various genes can occur at different time (upward and downward genes). Another extremity is when alteration if gene expression is buffered by other genes. In ontogenesis, this effect is called robustness, and its function is to protect ontogenetic pathways from environmental and genetic perturbances. I-2.12. Evolution of genetic regulation (16-17b. slides) Certain genes of higher-order organisms are more complex than the same in lower-order organisms. It means that the particular gene is composed of more functional domain in complex organism than in primitive ones. However, these alterations occur in only large evolutionary distances involving only a few gene. Much more frequent situation is that the same gene has the same function id distantly related species. There is not a big difference in the number of genes between human, fruit fly and C. elegans (a worm). Several developmental genes can be replaced between fruit fly and mouse without exerting observable changes (anatomical, behavioral, etc.). Thus, animals have the same toolkits (genes) with the 5 same function, what varies is the operation of genes in different species. Hat is, genes are expressed differently (different amount, space and time) in various organisms. The striping of zebra is a good example form the timing of gene expression. The number of stripes in the three zebra species is determined by the starting of striping during the embryogenesis. An example for the spatial alteration of gene expression is the loss of the legs of snakes. The Hoxc-6 and Hox-8 genes of snakes are expressed in the same segments of the snakes, which results in the development of vertebrates instead of legs. II. Regulation after transcription II-1. Posttranscriptional regulation II-1.1. mRNA processing (18. slide) The vast majority of eukaryotic mRNAs are subject to post-transcriptional processing. A typical pre-mRNA (it is a form of heteronuclear RNA coding for an mRNA) is composed of the following parts. Exon becomes the part of mRNA, while the introns are removed by splicing. The upstream non-coding sequence of the first exon is termed 5’-UTR (untranslated region) or leader sequence. The codon AUG coding for methionine is the translation start site of the mRNA. The downstream non-coding region of the last exon is termed trailer (or 3’UTR). The poly-adenylation signal is located at the downstream region of the 3’-UTR. II-1.2. Splicing (19. – 20. slides) Splicing is a modification of genetic information after transcription, in which introns are removed and exons are joined. Splicing is an essential process in eukaryotic pre-mRNA processing that must precede translation. The so-called spliceosomal introns are spliced by the spliceosome*, by means of a large RNA-protein complex composed of five small nuclear ribonucleoproteins (snRNPs (pronounced "snurps") and many accessory proteins. The splice donor site (contains a GU consensus sequence) is located at the 5’-end-, while splice acceptor site (contains a AG consensus sequence) at the 3’-end exon/intron boundary. Functions of introns: 1. Alternative splicing: more than one proteins can be produced form the same RNA. 2. It contains regulatory regions 3. Most of them are junks, that is, it does not perform any useful function neither for the gene, cell, or the organism. The genome simply is not able to get rid of them. 4. Introns have structural roles II-1.3. Capping (21. slide) Post-transcriptional processing of the 5' end of the RNA product of DNA transcription comes in the form of a process called capping. At the end of transcription, the 5' end of the RNA transcript contains a free triphosphate group since it was the first incorporated nucleotide in the chain. The capping process replaces the triphosphate group with another structure called the "cap", which is 7-methyl guanozine. The cap is added by the enzyme guanyl transferase. This enzyme catalyzes the reaction between the 5' end of the RNA transcript and a guanine triphosphate (GTP) molecule. In the case of alternative capping, depending on cell type, cap is formed at different position of the mRNA. Alternative capping is a relative rare form of posttranscriptional regulation. Functions of capping: 6 1. Protection against exonucleases 2. Needed for the binding of ribosomes II-1.4. Polyadenylation (22. slide) Polyadenylation is the covalent linkage of a polyadenylyl moiety to a mRNA molecule. In eukaryotic organisms, polyadenylation is the mechanism by which most mRNA molecules are terminated at their 3' ends. The poly A tail aids in mRNA stability by protecting it from exonucleases. Polyadenylation is also important for export of the mRNA from the nucleus, and for translation. Polyadenylation occurs during and immediately after transcription of DNA into RNA in the nucleus. After transcription has been terminated, the mRNA chain is cleaved through the action of an endonuclease complex associated with RNA polymerase. The cleavage site is characterized by the presence of the base sequence AAUAAA near the cleavage site. After the mRNA has been cleaved, 50 to 250 adenosine residues are added to the free 3' end at the cleavage site. This reaction is catalyzed by polyadenylate polymerase. Polyadenylation signal, cleavage site and site of polyA addition are distinct, but located close to each other. Functions: 1. Protection against exonucleases 2. Determination of life time II-1.4. mRNA editing (23. slide) RNA editing is a co- or post-transcriptional mechanism which alters the content of the mRNA. For example, in mammalian apolipoprotein mRNA one CU transition (substitution editing) by cytidine deaminase changes the CAA codon of the mRNA to UAA stop codon. This results in the generation of a truncated yet functional transcript in the intestine. In glutamate receptor expressing in several neuron types, the glutamate receptor mRNA is modified by RNA editing to contain different amino acid composition, which result in altered functioning of the resulting receptor protein. RNA editing occurs rarely in nature II.1.5. Alternative gene usage (24. – 26. slides) Alternative promoter usage means that a gene can be transcribed from various promoters in different tissues, resulting in proteins with varying length or with varying amino acid sequences if alternative splicing also occurs. Alternative polyadenylation results polypeptides with various length and exon content if it is coupled with alternative splicing. Approximately 60% of human genes encodes at least two splice variants. II-1.6. mRNA transport (27. slide) Eukaryotic mRNAs must leave the nucleus in order to be translated into proteins. Mature mRNAs exit through the nuclear pores, but the underlying mechanisms are not fully understood. A large portion of unprocessed transcripts never leave the nucleus and are degraded. II-1.7. mRNA localization (28. slide) Protein traveling to the appropriate organelles is directed by the signal peptides locating on the N-terminal of proteins. Another possibility for pass a certain protein to the desired organelle is based on mRNA targeting. Some mRNAs contain a zip code on the 5’ termini, which contains information for the subcellular targeting of mRNA. II-1.8. mRNA stability (29. slide) 7 The stability of mRNAs can vary to a great extent, which may change in response to regulatory signals. The following sequences and processes affect the mRNA half-life: AUrich elements, secondary structure, deadenylation of the poly(A) tail, 5’ de-capping and fragment degradation. II-2. Regulation of translation (30. slide) Translation can be regulated at every steps. The most important control point is the translation initiation. II-3. Posttranslational regulation II-3.1. Protein degradation (31. slide) Protein degradation in eukaryotes requires a protein cofactor called ubiquitin*, which, by binding to the proteins, identifies them for degradation by proteolytic enzymes. Specific amino acids at the N-termini of proteins determine the rate of ubiquitin binding and thus the stability of proteins. II-3.2. Protein processing and modification (32. slide) Protein degradation in eukaryotes requires a protein cofactor called ubiquitin*, which, by binding to the proteins, identifies them for degradation by proteolytic enzymes. Specific amino acids at the N-termini of proteins determine the rate of ubiquitin binding and thus the stability of proteins. III. Regulatory RNAs Although RNAs are best known for their roles in translating genetic information into proteins, the analyses of data on genomic sequences indicate the hitherto considerably underestimated importance of regulatory (noncoding) RNAs, including antisense RNAs* micro (mi)RNAs* and small interfering (si)RNAs*. Types of RNAs (33. slide) 8 RNA s Non-coding RNA Coding RNA Transcription RNA mRNA tRNA messenger transfer rRNA ribosomal Regulatory RNA siRNA aoRN miRN small interfering micro antisense overlapping snRNA snoRNA small nuclear small nucleolar III-1. Micro RNAs (34. - 35. slides) A continuously increasing number of miRNAs have been described in the genomes of several multicellular organisms. MicroRNA genes yield RNA transcripts that are processed into short single-stranded segments, which then double over on themselves to form hairpin structures. It has been proposed that they act as components of protein/RNA complexes. A miRNA can both pair exactly with a mRNA and cause its degradation via RNA interference* (RNAi; see bellow) or it can pair partially with a message and shut off translation. Recent studies involving computational approaches suggest that the human genome may encode well over 1500 different miRNAs; the number known is rising rapidly. MicroRNAs are promiscuous transactivators, i.e. a single RNA is assumed to regulate the expression of several genes. It is hypothesized that up to one-third of human genes are regulated by these small RNAs. A miRNA is a form of single-stranded (ss)RNS which is typically 20-25 nucleotides long. The miRNAs are transcribed from DNA, but are not translated into protein. The DNA sequence that codes for a miRNA gene is longer than the miRNA. This DNA sequence includes the miRNA sequence and an approximate reverse complement. When this DNA sequence is transcribed into a single-stranded RNA molecule, the miRNA sequence and its reversecomplement base pair to form a double stranded RNA hairpin loop; this forms a primary miRNA structure (pri-miRNA). Drosha, a nuclear enzyme, cleaves the base of the hairpin to form pre-miRNA. The pre-miRNA molecule is then actively transported out of the nucleus into the cytoplasm. The Dicer enzyme then cuts 20-25 nucleotides from the base of the hairpin to release the mature miRNA. The function of miRNAs appears to be in gene regulation. For that purpose, a miRNA is complementary to a part of one or more mRNAs, usually at a site in the 3’-UTR (untranslated region). The annealing of the miRNA to the mRNA inhibits protein translation. In some cases, the formation of the double-stranded RNA through the binding of the miRNA triggers the degradation of the mRNA transcript through a process similar to RNAi, though in other cases it is believed that the miRNA complex blocks 9 the protein translation machinery or otherwise prevents protein translation without causing the mRNA to be degraded. III-2. Overlapping RNAs (36. - 37. slides) Natural cis-encoded antisense RNAs are endogenous transcripts that are transcribed from the opposite strand of the same genomic locus as the sense RNA and have a region of perfect overlap with the sense transcripts. Very surprising novel data suggest that at least 30-40% of genes are under the control of cis-antisense RNAs. The binding of mRNAs and antisense transcripts can sterically block translation from mRNA or, alternatively, it may trigger the RNA interference pathway, which eventually leads to the degradation of mRNA. III-3. RNA interference (38. - 39. slides) RNA interference (RNAi) is a mechanism in molecular biology where the presence of certain fragments of double-stranded RNA (dsRNA) interferes with the expression of a particular gene. RNAi appears to be a highly potent and specific process which is actively carried out by special mechanisms in the cell, known as the RNA interference machinery. While the complete details of how it works are still unknown, it appears that the machinery, once it finds a double-stranded RNA molecule, cuts it up, separates the two strands, and then proceeds to destroy other single-stranded RNA molecules that are complementary to one of those segments. dsRNAs direct the creation of small interfering RNAs (siRNAs*) which target RNA-degrading enzymes (RNAses) to destroy transcripts complementary to the siRNAs. The genetic information of many viruses is held in the form of double-stranded RNA, so it is likely that the RNA interference machinery evolved as a defense against these viruses. The machinery is however also used by the cell itself to regulate gene activity: certain parts of the genome are transcribed into microRNAs, short RNA molecules that fold back on themselves in a hairpin shape to create a double strand. When the RNA interference machinery detects these double strands, it will also destroy all mRNAs that match the microRNA, thus preventing their translation and lowering the activity of many other genes. RNAi has been linked to various cellular processes, including the formation of centromeric structure and gene regulation, through microRNAs and heterochromatin formation. The effectiveness of RNAi lies in two processes: (1) Cleavage of mRNA by the RISC complex; (2) amplification of the original signal by RNA-dependent RNA polymerase by utilizing mRNA as a template and the complementary strand of siRNAs as a primer for the amplification step in the first part shows that transcription and translation, and the second part presents the mechanism of RNA interference. Together: DICER forms siRNAs from double-stranded RNA molecules. The fate of siRNAs can be as follows. 1. The siRNA bind to RISC, and after being single stranded, it lads the RISC to the target mRNA, where the RISC cut the mRNA at a single point, which is followed by the degradation of mRNA by Razes. 2. The single stranded siRNS detaches (or not attaches to) from the RISC, and binds directly to the mRNA. This binding is recognized by an RNA-dependent RNA polymerase, which utilizes the siRNA as a primer and the mRNA as a template for the synthesis of second RNA strand. The newly formed double-stranded RNA will serve as a substrate for DICER, which will create new and new siRNAs. This amplification step make RNA interference so effective. RNA interference participate in gene expression regulation as an antiviral defense mechanism. 10 Role in the medicine The double stranded (ds)RNAs that trigger RNAi may be usable as drugs. Another speculative use of dsRNA is in the repression of essential genes in eukaryotic human pathogens or viruses that are dissimilar from any human genes; this would be analogous to how existing drugs work. RNAi interferes with the translation process of gene expression and appears not to interact with the DNA itself. Proponents of therapies based on RNAi suggest that the lack of interaction with DNA may alleviate some patients' concerns about alteration of their DNA (as practiced in gene therapy), and suggest that this method of treatment would likely be no more feared than taking any prescription drug. For this reason RNAi and therapies based on RNAi have attracted much interest in the pharmaceutical and biotech industries. Glossary Chromatin is the complex of DNA and protein found inside the nuclei of eukaryotic cells. The major proteins involved in chromatin are histone proteins, although many other chromosomal proteins have prominent roles too. The functions of chromatin are to package DNA into a smaller volume to fit in the cell, to strengthen the DNA to allow mitosis and meiosis, and to serve as a mechanism to control expression. Changes in chromatin structure are affected mainly by methylation (DNA and proteins) and acetylation (proteins). Chromatin is easily visualized by staining, hence its name, which literally means colored material. Simplistically, there are three levels of chromatin organization: 1. DNA wrapping around nucleosomes - The "beads on a string" structure. 2. A 30 nm condensed chromatin fiber consisting of nucleosome arrays in their most compact form. 3. Higher level DNA packaging into the metaphase chromosome. DNA chips: Scientists use DNA microarrays to measure the expression levels of large numbers of genes simultaneously. Epigenetic inheritance: the same genetic content can determine more than one phenotype as a result of, for example, maternal effects. Exon shuffling: gaining novel domains of proteins by acquiring a new exon from another gene located at other part of the genome during evolution. Forward genetics: The experimental procedure that begins with a random mutation and a subsequent search for the altered phenotype and the mutant gene responsible for this phenotype. Functional genomics uses high-throughput techniques like DNA microarrays* and proteomics* to describe the function and interactions of genes. These techniques allow the analysis of the expression level of a huge number of gene at the same time. Gene expression is a multi-step process that begins with transcription, followed by post transcriptional modification and translation. Gene networks are genetic modules composed of functionally-linked genes, which determine the development and operation of particular traits, physiological processes or behaviors. 11 The genome of an organism is the whole hereditary information of an organism that is encoded in the DNA (or, for some viruses, RNA). This includes both the genes and the noncoding sequences. Genomics is the study of an organism's/species’ genome. A knockout animal is a genetically engineered animal (usually mouse) one or more of whose genes have been made inoperable. Knockout is a route to learning about a gene that has been sequenced (revealing the order of bases) but has an unknown or incompletely known function. Phenotype: anything that is part of the observable structure, function or behavior of an organism Protein chips (= protein microarrays) are measurement devices used in biomedical applications to determine the presence and/or amount (referred to as quantitation) of proteins in biological samples. Proteome The entirety of proteins in existence in an organism. Most importantly, while the genome* is a rather constant entity, the proteome differs from cell to cell and is constantly changing through its biochemical interactions with the genome and the environment. Proteomics is the large-scale study of proteins (simultaneous analysis of a large number of proteins). This term was coined to make an analogy with genomics*, and while it is often viewed as the "next step", proteomics is much more complicated than genomics. Regulatory RNAs A commonly used synonym is non-coding RNA, RNA molecules that function without being translated into proteins. Their functions include regulation of gene expression at the levels of transcription (chromatin modification) and translation. Reverse genetics The experimental approach that begins with a cloned segment of DNA, followed by the insertion of this DNA to the host genome. The foreign DNA can serve both a transgene, which is over-expressed in the host animal, or it can serve to knock out an endogenous gene of the host. The aim of reverse genetics to find altered phenotype resulted by the genetic manipulation. Ribonuclease (RNase) is an enzyme that catalyzes the breakdown of RNA into smaller components. Signal transduction: is any process by which a cell converts one kind of signal or stimulus into another. Processes referred to as signal transduction often involves a sequence of biochemical reactions inside the cell, which are carried out by enzymes and other proteins linked through second messengers. Silent codon positions. The genetic code is redundant, which means that in most cases more than one codon determines a single amino acids. Those base replacements, which do not result in the change of amino acids are called as silent changes. Those positions of codons (generally third ones), which contain the replaceable bases are called silent codon positions. Small interfering RNA (siRNA), are a class of 20-25 nucleotide-long RNA molecules that interfere with the expression of genes. They are naturally produced as part of the RNA 12 interference (RNAi) pathway by the enzyme Dicer. They can also be exogenously (artificially) introduced by investigators to bring about downregulation of a particular gene. SiRNA's have a well defined structure. Briefly, this is a short (usually 21-nucleotide) doublestrand of RNA (dsRNA) with 2-nucleotide overhangs on either end, including a 5' phosphate group and a 3' hydroxy (-OH) group. Spliceosome is a complex of RNA and many protein subunits that remove the non-coding introns from unprocessed mRNA. The RNAs that spliceosomes consist of are named U1, U2, U4, U5, and U6, and participate in several RNA-RNA and RNA-protein interactions. Transcription factor: a protein that binds DNA at a specific promoter and enhancer or other transcription factors, and thereby directly controls transcription. Transcription factors can be selectively activated or deactivated by other proteins, often as the final step in signal transduction*. Transgenic organism: An organism that has integrated foreign DNA into its germ line as a result of the experimental introduction of DNA. Recombinant DNA techniques are commonly used to produce a transgenic organism. Ubiquitin is a 76 residue polypeptide that can be conjugated to specific proteins by members of a complex family of enzyme cascade systems, whereby signaling that the particular protein is destined for degradation. 13