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1. Genes and RNA The initial products of all genes is a sequence of ribonucleic acid (RNA). RNA is produced by a process that copies the nucleotide sequence in DNA. Since this process is reminiscent of transcribing (copying) written words, the synthesis of RNA is called transcription. The DNA is said to be transcribed into RNA, and the RNA is called a transcript. One way to think about the different biological roles of DNA and RNA is to consider that the DNA (that is, the genome) is the instruction manual for producing all the RNAs that the cell needs, whereas RNA is the erasable readout of those parts of the manual relevant to any given task. Genetica per Scienze Naturali a.a. 03-04 prof S. Presciuttini 2. Properties of RNA Although RNA and DNA are both nucleic acids, RNA differs in several important ways: 1. RNA is a single-stranded nucleotide chain, not a double helix. One consequence of this is that RNA can form a much greater variety of complex three-dimensional molecular shapes than can double-stranded DNA. 2. RNA has ribose sugar in its nucleotides, rather than deoxyribose. As the names suggest, the two sugars differ in the presence or absence of just one oxygen atom. Analogous to the individual strands of DNA, there is a phosphate-ribose backbone to RNA, with a base covalently linked to the 1 position on each ribose. Genetica per Scienze Naturali a.a. 03-04 prof S. Presciuttini 3. Uracil instead of thymine The nucleotides of RNA carry the bases adenine, guanine, and cytosine, but the pyrimidine base uracil (abbreviated U) is found in place of thymine: However, uracil forms hydrogen bonds with adenine just as thymine does. Genetica per Scienze Naturali a.a. 03-04 prof S. Presciuttini 4. Classes of RNA RNAs can be grouped into two general classes: Some RNAs are intermediaries in the process of decoding genes into polypeptide chains; these molecules are called "informational" RNAs. In the other class, the RNA itself is the final, functional product. These RNAs are called "functional" RNAs Genetica per Scienze Naturali a.a. 03-04 prof S. Presciuttini 5. Informational RNAs For the vast majority of genes, the RNA is only an intermediate in the synthesis of the ultimate functional product, which is a protein. The informational RNA of this vast majority of genes is always messenger RNA (mRNA). In prokaryotes, the transcript, as it is synthesized directly from the DNA (the primary transcript), is the mRNA. In eukaryotes, however, the primary transcript is processed through modification of the 5’ and 3’ ends and removal of pieces of the primary transcript (introns). At the end of this pre-mRNA processing, an mRNA is produced. The sequence of nucleotides in mRNA is converted into the sequence of amino acids in a polypeptide chain by a process called translation. In this connection the word translation is used in much the same way as we use it in translating a foreign language: the cell has a way of translating the language of RNA into the language of polypeptides. Proteins are made up of one or more polypeptide chains. Genetica per Scienze Naturali a.a. 03-04 prof S. Presciuttini 6. Functional RNAs Functional RNAs action is purely at the level of the RNA; they are never translated into polypeptides. Each class of functional RNA is encoded by a relatively small number of genes (a few tens to a few hundred). The main classes of functional RNAs contribute to various steps in the informational processing of DNA to protein. Two classes of functional RNAs are found in all organisms: Transfer RNA (tRNA) molecules act as transporters that bring amino acids to the mRNA during the process of translation (protein synthesis). The tRNAs are general components of the translation machinery; they can bring amino acids to the mRNA of any protein-coding gene. Ribosomal RNAs (rRNAs) are components of ribosomes, which are large macromolecular assemblies that act as guides to coordinate the assembly of the amino acid chain of a protein. Ribosomes are composed of several types of rRNA and about 100 different proteins. As in the case of tRNA, the rRNAs are general translational components that can be used to translate the mRNA of any protein-coding gene. Genetica per Scienze Naturali a.a. 03-04 prof S. Presciuttini 7. One DNA strand is the template Transcription relies on the complementary pairing of bases. The two strands of the DNA double helix separate locally, and one of the separated strands acts as a template (alignment guide) for RNA synthesis. In the chromosome overall, both DNA strands are used as templates, but in any one gene only one strand is used, and in that gene it is always the same strand. One or the other DNA strand is used as transcriptional template. Genetica per Scienze Naturali a.a. 03-04 prof S. Presciuttini 8. 5’3’ RNA growth is always in the 5’3’ direction; in other words, nucleotides are always added at a 3’ growing tip: RNA polymerase moves always from the 3’ end of the template strand, creating an RNA strand that grows in a 5’3’ direction (since it must be antiparallel to the template strand). Some genes are transcribed from one strand of the DNA double helix; other genes use the other strand as the template Genetica per Scienze Naturali a.a. 03-04 prof S. Presciuttini 9. Transcription in action Transcription of ribosomal RNA (rRNA) genes in the developing egg cell of the spotted newt Eukaryotes have several hundred identical genes encoding ribosomal RNA. The long filaments are DNA molecules coated with proteins. The fibers extending in clusters from the main axes are molecules of ribosomal RNA which will be used in the construction of the cell's ribosomes. Transcription begins at one end of each gene, with the RNA molecules getting longer as they proceed toward completion. Note the large number (up to 100) of RNA molecules that are transcribed simultaneously from each gene. Genetica per Scienze Naturali a.a. 03-04 prof S. Presciuttini 10. RNA Polymerases In most prokaryotes, a single RNA polymerase does the job of transcribing all types of RNA. Eukaryotes have three different RNA polymerases, which specialize as follows: 1. RNA polymerase I (Pol I) transcribes rRNA genes. 2. RNA polymerase II (Pol II) transcribes protein-coding genes. 3. RNA polymerase III (Pol III) transcribes other functional RNA genes (for example, tRNA genes). In eukaryotes, transcription of nuclear chromosomes takes place entirely within the nucleus, and the transcripts then move through nuclear pores out into the cytoplasm, where translation occurs. Since prokaryotes have no nucleus, there is no comparable movement of transcripts, and translation can take place immediately, right on the growing transcript. Genetica per Scienze Naturali a.a. 03-04 prof S. Presciuttini 11. Three stages of transcription Transcription is usually described in terms of three distinct stages: Initiation Elongation Termination Genetica per Scienze Naturali a.a. 03-04 prof S. Presciuttini 12. INITIATION A DNA sequence to which RNA polymerase binds to initiate transcription is termed a promoter. A promoter is part of the regulatory region adjacent to the coding region of a gene. Since an RNA transcript is made in the 5’3’ direction, the convention is to view the gene in the 5’3’ orientation, too (the orientation of the nontemplate strand), even though transcription actually starts at the 3’ end of the template strand. By convention the first-transcribed end of the gene is called the 5’ end. Using this view, the promoter is at the beginning of the gene and, so, is said to be at the 5’ end of the gene, and the regulatory region is called the 5’ regulatory region Genetica per Scienze Naturali a.a. 03-04 prof S. Presciuttini 13. The promoter Promoter sites have regions of similar sequences, as indicated by the yellow region in the 13 different promoter sequences in E. coli. Spaces (dots) included to maximize homology at consensus sequences. The gene governed by each promoter sequence is indicated on the left. Numbering is given in terms of the number of bases before () or after (+) the RNA synthesis initiation point. Genetica per Scienze Naturali a.a. 03-04 prof S. Presciuttini 14. The TATA box Two regions of partial similarity appear in virtually all promoters. These regions have been termed the -35 (minus 35) and -10 regions because of their locations relative to the transcription initiation point. RNA polymerase scans the DNA for a promoter sequence, binds to the DNA at that point, then unwinds it and begins the synthesis of an RNA molecule at the transcriptional initiation site. Hence, we see that the principle of DNA binding is a result of interactions between the protein (here, the RNA polymerase) and a specific base sequence in the DNA. Genetica per Scienze Naturali a.a. 03-04 prof S. Presciuttini 15. RNA polymerase in bacteria Schematic diagram of prokaryotic RNA polymerase. The core enzyme contains two a polypeptides, one b polypeptide, and one b’ polypeptide. The addition of the s subunit allows initiation at promoter sites. Genetica per Scienze Naturali a.a. 03-04 prof S. Presciuttini 16. The s factor In order to recognize their promoters, bacterial RNA polymerase enzymes require a specialized subunit called the sigma factor (σ), which directly contacts the promoter sequence. The complex formed by the sigma subunit with the remaining polymerase core subunits constitutes the bacterial holoenzyme. Bacteria contain a variety of sigma factors that specifically recognize different promoter sequences. It is therefore the sigma factor that determines which genes are transcribed. All cells have a primary sigma factor, which directs transcription from the promoters of essential housekeeping genes, and a variable number of alternative sigma factors whose levels or activities are increased in response to specific signals. E. coli, a symbiotic bacterium leading a relatively sheltered life in the gut of other organisms, has only 7 sigma factors. Genetica per Scienze Naturali a.a. 03-04 prof S. Presciuttini 17. Structure of a bacterial RNA polymerase The structure of the T. aquaticus holoenzyme shows how three structural domains of the sigma subunit bind to the core enzyme in a position to recognize the promoter elements. The DNA is numbered relative to the transcription start site at +1. The σ2 domain recognizes the 10 region (red), while the σ3 domain binds to the flanking base pairs of the extended -10 region. The σ4 domain, which binds to the -35 element (red), is anchored to a flexible flap of the β subunit that may allow movement of the σ4 subunit to allow for different spacings between the -35 and -10 regions. Genetica per Scienze Naturali a.a. 03-04 prof S. Presciuttini 18. ELONGATION Shortly after initiating transcription, the sigma factor dissociates from the RNA polymerase, which moves along the DNA, maintaining a transcription "bubble" to expose the template strand, and catalyzes the 3’ elongation of the RNA strand. The polymerase compares free ribonucleotide triphosphates with the next exposed base on the DNA template and, if there is a complementary match, adds it to the chain. Genetica per Scienze Naturali a.a. 03-04 prof S. Presciuttini 19. TERMINATION Specific nucleotide sequences in the DNA act as signals for RNA chain termination. In the mechanism called direct termination, the termination signal consist of about 40 bp containing a GC-rich palindrome, followed by an oligo A region, which forms a local stem-loop structure in the RNA. The resulting double-stranded RNA section is called a hairpin loop. It is followed by the terminal run of U's that correspond to the A residues on the DNA template. This sequence disrupts the base pairing of newly synthesized RNA with the DNA template, forcing the RNA and the polymerase to fall off. Genetica per Scienze Naturali a.a. 03-04 prof S. Presciuttini 20. RNA Processing in Eukaryotes Transcription works in much the same way in eukaryotes as in prokaryotes; that is, there are specific promoter sequences to which the RNA polymerase binds, and the polymerase moves along the gene synthesizing RNA in the 5’3’ direction. Genetica per Scienze Naturali a.a. 03-04 prof S. Presciuttini 21. The RNA polymerase II holoenzyme Model of RNA Polymerase II Transcription Initiation Machinery.The machinery depicted here encompasses over 85 polypeptides in 10 (sub) complexes: core RNA polymerase II (RNAPII) consists of 12 subunits; TFIIH, 9 subunits; TFIIE, 2 subunits; TFIIF, 3 subunits; TFIIB, 1 subunit, TFIID, 14 subunits; core SRB/mediator, more than 16 subunits; Swi/Snf complex, 11 subunits; Srb10 kinase complex, 4 subunits; and SAGA, 13 subunits. Genetica per Scienze Naturali a.a. 03-04 prof S. Presciuttini 22. mRNA maturation In eukaryotes, the initial product of transcription, the primary RNA transcript, is processed in several ways before its transport to the cytosol. These processing steps are all performed by specific proteins that bind to the RNA. Until it reaches its final, mature form, the primary transcript is sometimes called pre-mRNA. First, during transcription, a cap consisting of a 7-methylguanosine residue is added to the 5’ end of the transcript, linked by a triphosphate bond. Then an AAUAAA sequence near the 3’ end is recognized by an enzyme that cuts off the end of the RNA approximately 20 bases farther down. At this time a stretch of 150 to 200 adenine nucleotides called a poly(A) tail is added at the cut 3’ end. Next, a crucial splicing step removes any introns from the RNA transcript, converting pre-mRNA into mature mRNA. Genetica per Scienze Naturali a.a. 03-04 prof S. Presciuttini 23. Mechanism of exon splicing For many eukaryotic genes the capped and tailed transcripts are shortened by the elimination of internal introns before transport into the cytoplasm. Exon-intron junctions of mRNAs show specific sequences that are highly conserved, i.e., they are the same in most introns in most species. There is a GU at the 5’ splice site of the intron and an AG at the 3’ splice site in virtually all cases examined ("the GU-AG rule") Consensus sequences of 5’ and 3’ splice junctions in eukaryotic mRNAs. Almost all introns begin with GU and end with AG. From the analysis of many exon intron boundaries, extended consensus sequences of preferred nucleotides at the 5’ and 3’ ends have been established. In addition to AG, other nucleotides just upstream of the 3 splice junction also are important for precise splicing. Genetica per Scienze Naturali a.a. 03-04 prof S. Presciuttini 24. The spliceosome Other less well conserved sequences are found flanking these. These common configurations of the pre-mRNA are recognized by small nuclear ribonucleoprotein particles, or snRNPs, which catalyze the cutting and splicing reactions. During the process of splicing, the snRNPs, the primary transcript, and associated factors all come together to form a high-molecular-weight (60S) ribonucleoprotein complex, called a spliceosome, which catalyzes the splicing reactions. Genetica per Scienze Naturali a.a. 03-04 prof S. Presciuttini 25. The translation At INITIATION, the ribosome recognizes the starting point in a segment of mRNA and binds a molecule of tRNA bearing a single amino acid. In all bacterial proteins, this first amino acid is Nformylmethionine. In ELONGATION, a second amino acid is linked to the first one. The ribosome then shifts its position on the mRNA molecule, and the elongation cycle is repeated. At TERMINATION, when the stop codon is reached, the chain of amino acids folds spontaneously to form a protein. Subsequently, the ribosome splits into its two subunits, which rejoin before a new segment of mRNA is translated. Genetica per Scienze Naturali a.a. 03-04 prof S. Presciuttini 26. Ribosomes are protein factories The meeting place for amino acid bound tRNAs and mRNA is the ribosome. Ribosomes are large macromolecular assemblies acting like complex subcellular machines. Each ribosome consists of a large and a small subunit, which shows slight differences between prokaryotes and eukaryotes. Each subunit in turn is composed of several rRNA types and as many as 50 proteins. Ribosomes contain specific sites that enable them to bind to the mRNA, the tRNAs, and other specific protein factors, all required for protein synthesis. The addition of a single amino acid to the growing polypeptide chain in the course of translation of mRNA Genetica per Scienze Naturali a.a. 03-04 prof S. Presciuttini 27. The tRNAs The structure of an alanine tRNA, showing the aminoacyl-tRNA binding to its correct codon in mRNA. Some nuclotides carry rare modified bases The basis for the specificity between codon and amino acid lies in the structure of transfer RNA (tRNA) molecules. A molecule of tRNA has a clover-leaf shape consisting of four double-helical stems and three single-stranded loops. The middle loop carries a nucleotide triplet called the anticodon, whose job it is to bind with a specific codon in the mRNA by specific RNA-toRNA base pairing. Since codons in mRNA are read in the 5 3direction, anticodons are oriented in the 3 5 direction. Each tRNA is specific for only one amino acid and carries that amino acid attached at its free 3 end. Amino acids are added to the tRNA by enzymes called aminoacyl-tRNA synthetases. Each amino acid has a specific synthetase that links it to only those tRNAs that recognize the codons for that amino acid. Genetica per Scienze Naturali a.a. 03-04 prof S. Presciuttini 28. Chain termination Three codons of the genetic code, UAG, UGA, and UAA, do not specify an amino acid. These are called stop codons or termination codons. They can be regarded as punctuation marks ending the message encoded in the mRNA. Stop codons often are called nonsense codons. The three stop codons are not recognized by a tRNA, but instead by protein factors called release factors. When the peptidyl-tRNA is in the P site, the release factors bind to the A site in response to the chain terminating codons. The polypeptide is then released from the P site, and the ribosomes dissociate into two subunits, ending translation. In one way of analyzing DNA sequences to look for potential genes, computers are programmed to look for open reading frames (ORFs), which are long DNA sequences beginning with an initiation codon (for example 5-ATG-3) and ending with one of the three stop codons. Genetica per Scienze Naturali a.a. 03-04 prof S. Presciuttini