Genetica per Scienze Naturali a.a prof S. Presciuttini 1. 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 prof S. Presciuttini 2. 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 prof S. Presciuttini 3. 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. 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 prof S. Presciuttini 4. 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 prof S. Presciuttini 5. 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. 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. 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. 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. Next, a crucial splicing step removes any introns from the RNA transcript, converting pre-mRNA into mature mRNA.

Genetica per Scienze Naturali a.a prof S. Presciuttini 6. 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. 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") 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 prof S. Presciuttini 7. 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. 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 prof S. Presciuttini 8. 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 N- formylmethionine. 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 prof S. Presciuttini 9. The ribosome

Genetica per Scienze Naturali a.a prof S. Presciuttini 10. Polyribosomes Clusters of ribosomes may sit on a mRNA and make proteins, each making a strand of polypeptides. These clusters are called polyribosomes. When they are free in the cytoplasm, they are called free polyribosomes (linked by the mRNA). Or, they may bind to rough endoplasmic reticulum. Ribosomes are visualized as small (20 X 30 nm) ribonucleoprotein particles. They are formed from two subunits. The two ribosomal subunits leave the nucleus separately through the nuclear pores. The pores are structured to allow transit of only the subunits. Whole ribosomes are formed outside in the cytoplasm. This prevents protein synthesis from occurring in the nucleus.

Genetica per Scienze Naturali a.a prof S. Presciuttini 9. 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. 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. 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. 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.