Chapter 17 The Gene-Protein Connection DNA contains the instructions for the building of proteins through the assembly of RNA molecules Transcription and Translation. DNA contains the instructions for the building of proteins through the assembly of RNA molecules Transcription and Translation. Therefore the DNA serves as a template to ensure the proper sequence of amino acids. Therefore the DNA serves as a template to ensure the proper sequence of amino acids. 1909 British physician Archibald Garrod was the first to suggest that genes dictate phenotypes through the catalysis of enzymes for specific reactions on the cell British physician Archibald Garrod was the first to suggest that genes dictate phenotypes through the catalysis of enzymes for specific reactions on the cell. Based his theory on disease alkaptonuria. Based his theory on disease alkaptonuria. In the 1930s Beadle and Tatum investigated this theory by studying Neurospora crassa, a common bread mold. In the 1930s Beadle and Tatum investigated this theory by studying Neurospora crassa, a common bread mold. One Gene One Polypeptide Hypothesis There was a debate on whether each gene was responsible for a particular enzyme or protien. There was a debate on whether each gene was responsible for a particular enzyme or protien. Not all proteins are enzymes and many proteins are constructed from multiple polypeptide chains. Each polypeptide is specified by its own gene. Not all proteins are enzymes and many proteins are constructed from multiple polypeptide chains. Each polypeptide is specified by its own gene. Beadle and Tatums research lead to the hypothesis that each gene codes for a particular polypeptide. Beadle and Tatums research lead to the hypothesis that each gene codes for a particular polypeptide. RNA Ribonucleic acid Ribonucleic acid Single stranded with a uracil base substitution for thymine Single stranded with a uracil base substitution for thymine Produced in the nucleus and moves out into the cytoplasm Produced in the nucleus and moves out into the cytoplasm Three types Three types mRNA, tRNA, rRNA Transcription / Translation Transcription is the synthesis of RNA under the direction of DNA. Transcription is the synthesis of RNA under the direction of DNA. Translation is the synthesis of polypeptides under the direction MRNA. Translation is the synthesis of polypeptides under the direction MRNA. Although prokaryotes and eukaryotes make proteins in a similar fashion, there are differences. Although prokaryotes and eukaryotes make proteins in a similar fashion, there are differences. Transcription During transcription the gene determines the sequence of bases along the length of the MRNA. During transcription the gene determines the sequence of bases along the length of the MRNA. Only one of the DNA strands is necessary for this step. Some genes use one strand while others chose another. Yet they use the same strand every time they are transcribed. Only one of the DNA strands is necessary for this step. Some genes use one strand while others chose another. Yet they use the same strand every time they are transcribed. Transcription occurs in the nucleus releasing a primary transcript. Transcription occurs in the nucleus releasing a primary transcript. Codons The DNA is transcribed in a series of non- overlapping triplet sequences called codons. The DNA is transcribed in a series of non- overlapping triplet sequences called codons. mRNA is complementary to DNA not identical. mRNA is complementary to DNA not identical. Transcription An RNA transcript is produced by this process. An RNA transcript is produced by this process. Requires an RNA polymerase which assembles in a 53 direction. Requires an RNA polymerase which assembles in a 53 direction. 3 steps 3 steps initiation initiation elongation elongation termination termination Promotor Region The sequence on the DNA where the RNAP attaches and initiates transcription. The sequence on the DNA where the RNAP attaches and initiates transcription. Located several dozen nucleotides upstream from the start point. Located several dozen nucleotides upstream from the start point. The promotor region includes: The promotor region includes: 1. start site (codon) 1. start site (codon) 2. TATA box 2. TATA box Transcription initiation A group of proteins referred to as transcription factors mediate the binding of the RNAP and the initiation of transcription. A group of proteins referred to as transcription factors mediate the binding of the RNAP and the initiation of transcription. The combination of transcription factors and RNAP transcription initiation complex The combination of transcription factors and RNAP transcription initiation complex Once this complex is firmly attached, the DNA strands unwind and the enzymes begin to transcribe. Once this complex is firmly attached, the DNA strands unwind and the enzymes begin to transcribe. Elongation RNA polymerase II moves along the DNA strand, unwinding the helix, exposing the bases 10 to 20 at a time. RNA polymerase II moves along the DNA strand, unwinding the helix, exposing the bases 10 to 20 at a time. The polymerase adds the nucleotides to the 3 end of the growing RNA molecule. The polymerase adds the nucleotides to the 3 end of the growing RNA molecule. A single gene can be transcribed by several polymerases allowing a large output of protein at a time. A single gene can be transcribed by several polymerases allowing a large output of protein at a time. Termination A terminator is a sequence on the DNA that signals the end of transcription in prokaryotic cells. A terminator is a sequence on the DNA that signals the end of transcription in prokaryotic cells. In eukaryotic cells, the polymerase continues to transcribe a sequence on the DNA until it reaches a polyadenylation signal sequence (AAUAAA) in the mRNA. In eukaryotic cells, the polymerase continues to transcribe a sequence on the DNA until it reaches a polyadenylation signal sequence (AAUAAA) in the mRNA. Then about nucleotides downstream from the AAUAAA the proteins associated with the growing transcript cut the trancript free from the polymerase releasing the pre-mRNA. Then about nucleotides downstream from the AAUAAA the proteins associated with the growing transcript cut the trancript free from the polymerase releasing the pre-mRNA. Modification of mRNA Most eukaryotic cells modify the pre-mRNA prior to dispatch from nucleus by altering both ends and cutting/splicing sections of the molecule. Most eukaryotic cells modify the pre-mRNA prior to dispatch from nucleus by altering both ends and cutting/splicing sections of the molecule. The 5 end (cap) is capped by a modified Guanine nucleotide after the transcription of the first nucleotides. The 5 end (cap) is capped by a modified Guanine nucleotide after the transcription of the first nucleotides. The 3 tail consists of adenine nucleotides poly (A) tail The 3 tail consists of adenine nucleotides poly (A) tail RNA Splicing The average DNA transcription unit is about 8000 nucleotides long, yet the average protein code is about 1200 nucleotides long. The average DNA transcription unit is about 8000 nucleotides long, yet the average protein code is about 1200 nucleotides long. The sequence is not continuous. The sequence is not continuous. Non-coding sections Introns Non-coding sections Introns Coding sections Exons Coding sections Exons RNA Splicing (con.) Particles called small nuclear ribonucleoproteins (snRNPs) recognize specific cleaving sites located at the end of the introns. Particles called small nuclear ribonucleoproteins (snRNPs) recognize specific cleaving sites located at the end of the introns. A large group of snRNPs join together forming Splicesome. A large group of snRNPs join together forming Splicesome. Splicesomes interact with specific sites on the introns releasing it and then joining together the exons that flank the released intron. Splicesomes interact with specific sites on the introns releasing it and then joining together the exons that flank the released intron. Evolutionary Significance One idea is that introns have a regulatory role in the cell and that the splicing is necessary for the passing into the cytoplasm. One idea is that introns have a regulatory role in the cell and that the splicing is necessary for the passing into the cytoplasm. Alternative RNA splicing allows a gene to give rise to more than one polypeptide, depending on the segments treated as an exon. Alternative RNA splicing allows a gene to give rise to more than one polypeptide, depending on the segments treated as an exon. The presence of introns may facilitate the formation of a new, useful gene exon shuffling The presence of introns may facilitate the formation of a new, useful gene exon shuffling Providing more terrain for beneficial crossing over between exons, not to mention reshuffling during sexual reproduction. Providing more terrain for beneficial crossing over between exons, not to mention reshuffling during sexual reproduction. Translation In this process the information coded into a mRNA transcript is converted into a particular polypeptide sequence at the ribosome. In this process the information coded into a mRNA transcript is converted into a particular polypeptide sequence at the ribosome. tRNA acts to transfer amino acids from the cytoplasm to a ribosome. tRNA acts to transfer amino acids from the cytoplasm to a ribosome. tRNA are transcribed from the DNA template. They are used repeatedly. tRNA are transcribed from the DNA template. They are used repeatedly. Aminoacyl-tRNA synthetase is responsible for joining the correct amino acid to its tRNA. Aminoacyl-tRNA synthetase is responsible for joining the correct amino acid to its tRNA. There are 20 different aminoacyl synthetases each with a specific active site for a particular amino acid. There are 20 different aminoacyl synthetases each with a specific active site for a particular amino acid. The synthetase catalyzes the covalent attachment of the amino acid via an ATP molecule. The synthetase catalyzes the covalent attachment of the amino acid via an ATP molecule. The recognition of the mRNA by the tRNA is more relaxed due to the wooble affect. The recognition of the mRNA by the tRNA is more relaxed due to the wooble affect. Ribosomes Ribosomes are made within the nucleolus of rRNA and proteins imported from the cytoplasm into subunits that are then released to the cytoplasm. Ribosomes are made within the nucleolus of rRNA and proteins imported from the cytoplasm into subunits that are then released to the cytoplasm. Ribosome function to bring mRNA together with tRNA. Ribosome function to bring mRNA together with tRNA. 3 sites on the ribosome aid in the translation process. 1. P site (peptidyl) holds the tRNA carrying the growing chain. 2. A site (aminoacyl-tRNA) hold the tRNA carrying the next amino acid to be added to the chain. 3. E site (exit site) discharged tRNA leave the ribosome at this point. Translation Initiation A small ribosomal subunit binds to mRNA and an initiator tRNA that carries the amino acid methione. A small ribosomal subunit binds to mRNA and an initiator tRNA that carries the amino acid methione. The small subunit then scans the mRNA downstream until reaches a start codon where the initiator tRNA then hydrogen binds with the start codon. The small subunit then scans the mRNA downstream until reaches a start codon where the initiator tRNA then hydrogen binds with the start codon. At this point, the larger subunit binds with the complex forming the translation initiator complex. At this point, the larger subunit binds with the complex forming the translation initiator complex. Initiator proteins and GTP molecules are required for the making of this complex. Initiator proteins and GTP molecules are required for the making of this complex. Elongation Elongation factor proteins are needed for each addition of amino acid to the carboxyl end. Elongation factor proteins are needed for each addition of amino acid to the carboxyl end. The mRNA moves through the ribosome in a 5-3 direction. The ribosome moves with each codon uni-directionally. The mRNA moves through the ribosome in a 5-3 direction. The ribosome moves with each codon uni-directionally. Termination Elongation continues until a stop codon reaches the A site in the ribosome. Elongation continues until a stop codon reaches the A site in the ribosome. UGA, UAA, and UAG serve as stop codons. UGA, UAA, and UAG serve as stop codons. A release factor protein binds to the stop codon in the A site, causing the addition of a water molecule instead of an amino acid. A release factor protein binds to the stop codon in the A site, causing the addition of a water molecule instead of an amino acid. This reaction releases the polypeptide from the P site and exits the ribosome. This reaction releases the polypeptide from the P site and exits the ribosome. Modification of polypeptide During synthesis, a polypeptide chain begins to coil spontaneously forming a 3D conformational, functional protien. During synthesis, a polypeptide chain begins to coil spontaneously forming a 3D conformational, functional protien. In many cases, a chaperone protein is needed. In many cases, a chaperone protein is needed. Post-translational steps may include; attachments of sugars, lipids or phosphate groups, cleavage into 2 or more pieces or bonding of separate subunits together. Post-translational steps may include; attachments of sugars, lipids or phosphate groups, cleavage into 2 or more pieces or bonding of separate subunits together. All translation begins on a free floating ribosome. All translation begins on a free floating ribosome. The polypeptides destined for the endomembrane system or secretion are marked by a signal peptide which targets the protein to the ER. The polypeptides destined for the endomembrane system or secretion are marked by a signal peptide which targets the protein to the ER. Signal recognition particle complex recognizes the signal peptide bringing the ribosome to a receptor protein built into the ER membrane. Signal recognition particle complex recognizes the signal peptide bringing the ribosome to a receptor protein built into the ER membrane. The signal peptide is removed by an enzyme. The signal peptide is removed by an enzyme. Other signal peptides are used to target the mitochondria, chloroplasts and nucleus. Other signal peptides are used to target the mitochondria, chloroplasts and nucleus. Prokaryotes/Eukaryotes Polymerases differ Polymerases differ Eukayotes use transcription factors Eukayotes use transcription factors Compartmentalization Compartmentalization Processing of polypeptide Processing of polypeptide Use of complicated targeting proteins Use of complicated targeting proteins Base-pair substitution In some cases there can be silent mutations due to redundancy of the genetic code. In some cases there can be silent mutations due to redundancy of the genetic code. May change the amino acid but have little effect on the protein. May change the amino acid but have little effect on the protein. If the substitution causes a detectable change in the protein its function will be altered. If the substitution causes a detectable change in the protein its function will be altered. Substitution mutations are usually missense, where the altered codon still codes for an amino acid that makes sense but not the right sense. Substitution mutations are usually missense, where the altered codon still codes for an amino acid that makes sense but not the right sense. Nonsense mutation can occur when the substitution changes the amino acid into a stop codon causing translation to be stopped prematurely. Nonsense mutation can occur when the substitution changes the amino acid into a stop codon causing translation to be stopped prematurely. Base-pair Insertions/Deletions These mutations have a disastrous effect on the resultant protein. These mutations have a disastrous effect on the resultant protein. An insertion/deletion may alter the reading frame. This is termed a frameshift mutation. An insertion/deletion may alter the reading frame. This is termed a frameshift mutation. Unless the frameshift occurs near the end of the gene, it will be nonfunctional. Unless the frameshift occurs near the end of the gene, it will be nonfunctional.