Genetica per Scienze Naturali a.a prof S. Presciuttini 1. Genes and RNA The initial products of all genes is a sequence of ribonucleic acid (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. 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. 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. 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 prof S. Presciuttini 2. Properties of RNA Although RNA and DNA are both nucleic acids, RNA differs in several important ways: 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 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: 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. However, uracil forms hydrogen bonds with adenine just as thymine does.

Genetica per Scienze Naturali a.a prof S. Presciuttini 4. Classes of RNA RNAs can be grouped into two general classes: 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 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). 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. 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 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: 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 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. 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 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 prof S. Presciuttini 9. Transcription in action 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. Transcription of ribosomal RNA (rRNA) genes in the developing egg cell of the spotted newt

Genetica per Scienze Naturali a.a prof S. Presciuttini 10. RNA Polymerases In most prokaryotes, a single RNA polymerase does the job of transcribing all types of RNA. 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: 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. 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 prof S. Presciuttini 11. Three stages of transcription Transcription is usually described in terms of three distinct stages: Transcription is usually described in terms of three distinct stages:  Initiation  Elongation  Termination

Genetica per Scienze Naturali a.a 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 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 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 prof S. Presciuttini 15. RNA polymerase in bacteria Schematic diagram of prokaryotic RNA polymerase. The core enzyme contains two  polypeptides, one  polypeptide, and one  ’ polypeptide. The addition of the  subunit allows initiation at promoter sites.

Genetica per Scienze Naturali a.a prof S. Presciuttini 16. The  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. 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. 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. 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 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 σ 2 domain recognizes the - 10 region (red), while the σ 3 domain binds to the flanking base pairs of the extended -10 region. The σ 4 domain, which binds to the -35 element (red), is anchored to a flexible flap of the β subunit that may allow movement of the σ 4 subunit to allow for different spacings between the -35 and -10 regions.