Chapter 4: Flow of Genetic Information (Part 2) PDF

Chapter 4: Flow of genetic information (Part 2) Structure of one chain of DNA Simple and elegant Double helix structure Other structures (Bacterial DN...

Chapter 4: Flow of genetic information (Part 2) Structure of one chain of DNA Simple and elegant Double helix structure Other structures (Bacterial DNA, RNA) DNA replication Function Splicing Transcription Translation Splicing Because of the base pairing rules, the sequence of one strand determines the sequence of the partner strand. The two strands can be separated and complementary sequences synthesized to generate two identical daughter strands. Because the two daughter helices have one parent strand and one newly synthesized strand, the replication process is called semiconservative replication.  Meselson and Stahl elegantly demonstrated (in 1958) that replication is semiconservative by growing bacteria in growth media supplemented with 15N. The bacteria were then shifted to growth media with 14N as the nitrogen source. Density gradient centrifugation established that upon the shift to 14N medium, newly synthesized DNA consisted of DNA with equal parts 15N-DNA and 14N-DNA, a result consistent with semiconservative replication. N14 lighter and common, N15 heavier Slideshare.net  Resolution of 14N DNA and 15N DNA by density-gradient centrifugation (A) Ultraviolet-absorption photograph of a centrifuged cell showing the two distinct bands of DNA. (B) Densitometric tracing of the absorption photograph. N14 lighter and common, N15 heavier  Meselson and Stahl Experiment: Three postulated methods of DNA replication Pathwayz.org Meselson and Stahl (concept) Quora.com Diagram of semiconservative replication Detection of semiconservative replication of E. coli DNA by density-gradient centrifugation The position of a band of DNA depends on its content of 14N and 15N. After 1.0 generation, all of the DNA molecules were hybrids containing equal amounts of 14N and 15N. After two generations, there were equal amounts of two bands of DNA. One was hybrid DNA, and the other was 14 N DNA. Meselson and Stahl concluded from these incisive experiments that replication was semiconservative, and so each new double helix contains a parent strand and a newly synthesized strand. Their results agreed perfectly with the Watson–Crick model for DNA replication 900-word paper, published in Nature, concluded, famously, "It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material." Published in Nature, April 25, 1953 titled “ Molecular structure of nucleic acids: Structure for deoxyribose nucleic acid” pg 737-738 Diagram of semiconservative replication After 1.0 generation, all of the DNA molecules were hybrids containing equal amounts of 14N and 15N. Meselson and Stahl concluded from these incisive experiments that replication was semiconservative, and so each new double helix contains a parent strand and a newly synthesized strand. Their results agreed perfectly with the Watson–Crick model for DNA replication During replication or transcription, the two strands of the DNA double helix must be separated. In the laboratory, DNA strands can be separated by heating a solution of DNA, a process called denaturation or melting. The temperature at which half of the DNA molecules are denatured is called the melting temperature (Tm). Melting can be observed because bases stacked in a double helix absorb less ultraviolet light than bases in a single-stranded molecule, a phenomenon called hypochromism. Upon cooling, the two strands can bind to one another to reform the double helix, a process called reannealing. Hydrogen bonds Covalent Bonds Glycosidic linkage  Hypochromism (A) Single-stranded DNA absorbs light more effectively than does double-helical DNA. (B) The absorbance of a DNA solution at a wavelength of 260 nm increases when the double helix is melted into single strands. During replication or transcription, the two strands of the DNA double helix must be separated. In the laboratory, DNA strands can be separated by heating a solution of DNA, a process called denaturation or melting. The temperature at which half of the DNA molecules are denatured is called the melting temperature (Tm). Melting can be observed because bases stacked in a double helix absorb less ultraviolet light than bases in a single-stranded molecule, a phenomenon called hypochromism. Upon cooling, the two strands can bind to one another to reform the double helix, a process called reannealing.  Structure of one chain of DNA Simple and elegant Double helix structure Other structures (Bacterial DNA, RNA) DNA replication Function Splicing Transcription Translation Splicing The flow of genetic information is from DNA to RNA to proteins, a scheme called the central dogma. DNA replication in E. coli requires more than 20 proteins, five of which, called DNA polymerases, catalyze the synthesis of new DNA.  The reaction catalyzed by DNA polymerase is: Key characteristics of DNA synthesis are: 1. Four deoxynucleoside triphosphates and Mg2+ are required. 2. A template strand is used to direct DNA synthesis. 3. A primer from which the new strand grows must be present. 4. Many DNA polymerases have nuclease activity that allows for the removal of mismatched bases. Polymerization reaction catalyzed by DNA polymerases Key characteristics of DNA synthesis are: 1. Four deoxynucleoside triphosphates and Mg2+ are required. 2. A template strand is used to direct DNA synthesis. 3. A primer from which the new strand grows must be present. A primer strand having a free 3’OH group must be already bound to the template strand. (Note: RNA primes the synthesis of DNA. RNA polymerase called primase synthesizes a short stretch of RNA-about five nucleotides-that is complementary to one of the template DNA strands. Chapter 28- Eight edition) 4. Many DNA polymerases have nuclease activity that allows for the removal of mismatched bases.  Strand-elongation reaction. DNA polymerases catalyze the formation of a phosphodiester bridge. Xlink.rsc.org The strand elongation is a nucleophilic attack by the 3’OH end of the growing strand on the innermost phosphorous atom of the deoxyribose nucleoside triphosphate A phosphodiester bridge is formed, and a pyrophosphate is released. The subsequent hydrolysis of pyrophosphate to yield two ions of orthophosphate (Pi) by pyrophosphatase drives the polymerization forward.  Strand-elongation reaction. DNA polymerases catalyze the formation of a phosphodiester bridge. The strand elongation is a nucleophilic attack by the 3’OH end of the growing strand on the innermost phosphorous atom of the deoxynucleoside triphosphate A phosphodiester bridge is formed, and a pyrophosphate is released. The subsequent hydrolysis of pyrophosphate to yield two ions of orthophosphate (Pi) by pyrophosphatase drives the polymerization forward.  e e e https://www.differencebetween.com The flow of genetic information is from DNA to RNA to proteins, a scheme called the central dogma. Exceptions (modifications of the existing machinery)!!!  Some viruses, such as the tobacco mosaic virus, have RNA genomes that are replicated by RNA-directed RNA polymerases. Retroviruses, such as HIV-1, have single-stranded RNA genomes that are converted into DNA double helices by the action of reverse transcriptase. Exceptions (modifications of the existing machinery)!!! https://www.sciencedirect.com  SARS-CoV-2 The coronavirus SARS-CoV-2, the cause of the COVID-19 pandemic https://www.sciencedirect.com Coronaviruses are a group of related RNA viruses that cause diseases in mammals and birds. In humans and birds, they cause respiratory tract infections that can range from mild to lethal. Inside the envelope of the coronavirus SARS-CoV-2, there is the nucleocapsid, which is formed from multiple copies of the nucleocapsid (N) protein, which are bound to the positive-sense single-stranded RNA genome in a continuous beads-on-a-string type conformation. The lipid bilayer envelope, membrane proteins, and nucleocapsid protect the virus when it is outside the host cell. Wikipedia.org  Some viruses, such as the tobacco mosaic virus, have RNA genomes that are replicated by RNA-directed RNA polymerases. Retroviruses, such as HIV-1, have single-stranded RNA genomes that are converted into DNA double helices by the action of reverse transcriptase. Exceptions (modifications of the existing machinery)!!! https://www.sciencedirect.com  Flow of information from RNA to DNA in retroviruses (HIV). Genes of some viruses are made of RNA Exceptions (modifications of the existing machinery)!!! Some viruses, such as the tobacco mosaic virus, have RNA genomes that are replicated by RNA-directed RNA polymerases. Retroviruses, such as HIV-1, have single-stranded RNA genomes that are converted into DNA double helices by the action of reverse transcriptase. Reverse transcriptase possesses several activities and catalyzes the synthesis of a complementary DNA strand, the digestion of the RNA, and the subsequent synthesis of the DNA strand.  Structure of one chain of DNA Simple and elegant Double helix structure Other structures (Bacterial DNA, RNA) DNA replication Function Splicing Transcription Translation Splicing The flow of genetic information is from DNA to RNA to proteins, a scheme called the central dogma. Other functions There are many species of RNA that perform a variety of functions. However, the three most abundant classes of RNA are ribosomal RNA (rRNA), messenger RNA (mRNA) and transfer RNA (tRNA). Messenger RNA is the template for protein synthesis or translation (Prokaryotes- one mRNA molecule for group of genes vs Eukaryotes- each gene) Transfer RNA (tRNA) carries amino acids in an activated form to the ribosome for peptide- bond formation, in a sequence dictated by the mRNA template. Consists of about 75 nucleotides. There are 20 amino acids; so how many tRNAs are required for translation at the minimum? Ribosomal RNA (rRNA) is the major component of ribosomes. In prokaryotes three kinds of rRNA called 23S, 16S, and 5S RNA because of their sedimentation behavior. One molecule of each of these species of rRNA is present in each ribosome. rRNA is the actual catalyst for protein synthesis. Most abundant S is Svedberg unit; bigger particles have higher S values The synthesis of RNA from a DNA template is called transcription, a process catalyzed by RNA polymerase. RNA polymerase has the following requirements: 1. A template. The sequence of the newly-synthesized RNA is complementary to the DNA template. The DNA strand that has the same sequence as the RNA product (with T instead of U) is called the coding strand. 2. Activated precursors in the form of the four ribonucleoside triphosphates. 3. Divalent metal ions, usually Mg2+ or Mn2+ RNA polymerase initiates and elongates the RNA product, with the chain growing in the 5 to 3 direction. The 3 OH of the growing chain attacks the inner most phosphoryl (α) group of the incoming ribonucleoside triphosphate.  RNA Polymerase This large enzyme comprises many subunits, including β (red) and β’ (yellow), which form a “claw” that holds the DNA to be transcribed. Notice that the active site includes a Mg2+ion (green) at the center of the structure. The curved tubes making up the protein in the image represent the backbone of the polypeptide chain. No primer required. Reminder: For DNA polymerase, RNA primes the synthesis of DNA. RNA polymerase called primase synthesizes a short stretch of RNA-about five nucleotides-that is complementary to one of the template DNA strands. Chapter 28- Eight edition  Strand-elongation reaction. RNA polymerases catalyze the formation of a phosphodiester bridge. RNA U U RNA U Xlink.rsc.org The strand elongation is a nucleophilic attack by the 3’OH end of the growing strand on the innermost phosphorous atom of the ribonucleoside triphosphate A phosphodiester bridge is formed, and a pyrophosphate is released. The subsequent hydrolysis of pyrophosphate to yield two ions of orthophosphate (Pi) by pyrophposphatase drives the polymerization forward. Transcription mechanism of the chain-elongation reaction catalyzed by RNA polymerase Differences from DNA polymerase: Does not require a primer for elongation. Does not correct mistakes as extensively as DNA polymerase Newly synthesized RNA has a base sequence complementary to that of the template strand of the DNA.  Complementarity between mRNA and DNA The base sequence of mRNA (red) is the complement of that of the DNA template strand (blue). The sequence shown here is from the tryptophan operon, a segment of DNA containing the genes for five enzymes that catalyze the synthesis of tryptophan. The other strand of DNA (black) is called the coding strand because it has the same sequence as the RNA transcript except for thymine (T) in place of uracil (U). Promoters are specific DNA sequences that direct RNA polymerase to the proper initiation site. There are often variations in the sequence of a promoter for different genes. The average of such variation is called the consensus sequence. Promoter sites for transcription in (A) prokaryotes and (B) eukaryotes Promoters are specific DNA sequences that direct RNA polymerase to the proper initiation site. Consensus sequences are shown. The first nucleotide to be transcribed is numbered +1. The adjacent nucleotide on the 5 side is numbered -1. The sequences shown are those of the coding strand of DNA. Elongation continues until a termination signal is detected. The simplest stop signal is the transcribed product of a segment of palindromic DNA. The RNA complement of the DNA stop signal forms a hairpin structure, followed by several uracil residues. Upon synthesis of the hairpin, the polymerase stalls, the RNA product is released and the DNA double helix reforms. In other cases, the protein rho is required for transcription termination. Stop signal: Base sequence of the 3’ end of an mRNA transcript in E. coli. (bacteria) A stable hairpin structure is followed by a sequence of uridine (U) residues. Elongation continues until a termination signal is detected. The simplest stop signal is the transcribed product of a segment of palindromic DNA. The RNA complement of the DNA stop signal forms a hairpin structure, followed by several uracil residues. Upon synthesis of the hairpin, the polymerase stalls, the RNA product is released and the DNA double helix reforms. In other cases, the protein rho is required for transcription termination. Less is known about the termination of transcription in eukaryotes. The important point now is that discrete start and stop signals for transcription are encoded in the DNA template. Less is known about the termination of transcription in eukaryotes. The important point now is that discrete start and stop signals for transcription are encoded in the DNA template. In eukaryotes, the 5 end of mRNA is modified by the attachment of a cap structure while the 3 end acquires a poly(A) tail. Modification of mRNA. Messenger RNA in eukaryotes is modified after transcription. A nucleotide “cap” structure is added to the 5 end, and a poly(A) tail is added at the 3 end. In eukaryotes, the 5 end of mRNA is modified by the attachment of a cap structure while the 3 end acquires a poly(A) tail. A “cap” structure, a guanosine nucleotide attached to the mRNA with an unusual 5’-5’ triphosphate linkage, is attached to the 5’end.  A “cap” structure, a guanosine nucleotide attached to the mRNA with an unusual 5’-5’ triphosphate linkage (is attached to the 5’end) The cap is added by the enzyme guanyl transferase. https://en.wikipedia.org/ The relative sizes of a globular protein and the mRNA that codes for it Has other functions too http://book.bionumbers.org  Structure of one chain of DNA Simple and elegant Double helix structure Other structures (Bacterial DNA, RNA) DNA replication Function Splicing Transcription Translation Splicing The flow of genetic information is from DNA to RNA to proteins, a scheme called the central dogma. Other functions  Reminder from the first lecture: Macromolecules in living cells Only six elements carbon, hydrogen, oxygen, nitrogen, phosphorus, and sulfur (CHNOPS) make up about 98% of the mass of all living organisms. The functions of macromolecules are directly related to their shapes and to the chemical properties of their monomers (same way that the arrangement of the letters in a word determine its sound and meaning.) http://www.contexo.info In 1958, Francis Crick wrote: “RNA presents mainly a sequence of sites where hydrogen bonding could occur. One would expect, therefore, that whatever went onto the template in a specific way did so by forming hydrogen bonds. It is therefore a natural hypothesis that the amino acid is carried to the template by an adaptor molecule, and that the adaptor is the part that actually fits onto the RNA. In its simplest form, one would require twenty adaptors, one for each amino acid.” Transfer RNA molecules react with specific amino acids in a reaction catalyzed by aminoacyl-tRNA synthetases. Transfer RNA molecules also contain a template recognition site, called the anticodon. The anticodon, which consists of three bases, recognizes a complementary 3 base sequence in the mRNA called the codon.  General structure of an aminoacyl-tRNA. The amino acid is attached at the 3 end of the RNA. Notice that the tRNA has a cloverleaf structure with many hydrogen bonds (green dots) between bases. Transfer RNA molecules also contain a template recognition site, called the anticodon. The anticodon, which consists of three bases, recognizes a complementary 3 base sequence in the mRNA called the codon.  Attachment of an amino acid to a tRNA molecule. RNA presents mainly a sequence of sites where hydrogen bonding could occur Contain an amino acid attachment site and a template- recognition site. Transfer RNA molecules react with specific amino acids in a reaction catalyzed by aminoacyl-tRNA synthetases. The amino acid (shown in blue) is esterified to the 3’-hydroxyl group of the terminal adenylate of tRNA. (In chemistry, an ester is a chemical compound derived from an acid in which at least one –OH group is replaced by an –O–alkyl group.) Wikipedia: Amino acid activation (also known as aminoacylation or tRNA charging) refers to the attachment of an amino acid to its Transfer RNA (tRNA). Aminoacyl transferase binds Adenosine triphosphate (ATP) to amino acid, PP is released. Each aminoacyl-tRNA synthetase is highly specific for a given amino acid. (Chapter 30 in eight edition- protein synthesis) Protein synthesis is a process of translation. Nucleic acid sequence information is translated into amino acid sequence information. The genetic code links these two types of information. Characteristics of the genetic code are: 1. Three nucleotides, called a codon, encode an amino acid. (20 amino acids, only four bases. Calculations show that a minimum of three bases are required to encode at least 20 amino acids. Experiments confirmed this. 2. The code is nonoverlapping. (e.g., ABC DEF) 3. The code has no punctuation. Read sequentially from a fixed starting point. 4. The code has directionality. It is read from the 5 end of the mRNA to the 3 end. 5. The code is degenerate in that some amino acids are encoded by more than one codon. WHY?  Translation initiation Bio.libretexts.org Translation Central dogma wikipedia xurl.pl  Translation initiation Bio.libretexts.org Translation elongation and The formation of the peptide bond, one of the formation of the peptide bond most important reactions in life, is a thermodynamically spontaneous reaction catalyzed by a site on the 23S rRNA of the 50S subunit called the peptidyl transferase center. This catalytic center is located deep in the 50S subunit near the tunnel that allows the nascent peptide to leave the ribosome. wikipedia Brooklyncuny.edu Most amino acids are encoded by more than one codon, a property called degeneracy. Degeneracy minimizes the deleterious effects of mutations. Most amino acids are encoded by more than one codon, a property called degeneracy. Only tryptophan and methionine are encoded by just one triplet each. Total: 64 codons Codons that specify the same amino acid are called synonyms- most synonyms differ only in the last base. (Dr. Anton Komar, BGES, CSU) Degeneracy minimizes the deleterious effects of mutations. Most organisms use the same genetic code. However, some organisms have slight modifications. For instance, in ciliated protozoa, codons that are stop signals in most organisms encode amino acids. Mitochondria also use variations of the genetic code. Messenger RNA is translated on ribosomes. The first codon is almost always AUG, which codes for methionine. In prokaryotes, the AUG is preceded by a purine rich sequence called the Shine-Dalgarno sequence. Formyl-Met-tRNA binds to the initiator codon. In eukaryotes, the AUG nearest the 5 end is the initiator codon. Location of the initiator codon establishes the reading frame. Initiation of protein synthesis. Start signals are required for the initiation of protein synthesis. Messenger RNA is translated on ribosomes. The first codon is almost always AUG, which codes for methionine. In prokaryotes, the AUG is preceded by a purine rich sequence called the Shine-Dalgarno sequence. Formyl-Met-tRNA binds to the initiator codon. In eukaryotes, the AUG nearest the 5 end is the initiator codon. Location of the initiator codon establishes the reading frame.  Reminder: A “cap” structure, a guanosine nucleotide attached to the mRNA with an unusual 5’-5’ triphosphate linkage (is attached to the 5’end) The cap is added by the enzyme guanyl transferase. https://en.wikipedia.org/  Translation initiation and termination As already mentioned, UAA, UAG, and UGA designate chain termination These codons are read not by tRNA molecules but rather by specific proteins called release factors (Section 30.3). Binding of a release factor to the ribosome releases the newly synthesized protein. Bio.libretexts.org Translation Central dogma wikipedia xurl.pl The flow of genetic information is from DNA to RNA to proteins, a scheme called the central dogma. Has other functions too- Eg., Splicing Exceptions to central dogma: Viruses  Structure of one chain of DNA Simple and elegant Double helix structure Other structures (Bacterial DNA, RNA) DNA replication Function Splicing Transcription Translation Splicing Eukaryotic genes are discontinuous, with coding regions called exons, interrupted by noncoding regions called introns. Introns were initially detected by electron microscopy studies. The gene for the β-chain of human hemoglobin has 3 exons and two introns. Structure of the β-globin gene Pre-messenger RNA contains exons and intron. It is first modified by the addition of a 5 cap and a poly A tail at the 3 end. The introns are spliced out to generate mature mRNA by large complexes called spliceosomes. Introns almost always begin with a GU and end with an AG. Transcription and post transcriptional processing of the β-globin gene. Pre-messenger RNA contains exons and intron. It is first modified by the addition of a 5’ cap and a poly A tail at the 3’ end. Splicing is a complex operation that is carried out by spliceosomes, which are assemblies of proteins and small RNA molecules (snRNA). The introns are spliced out to generate mature mRNA by large complexes called spliceosomes. snRNA plays the catalytic role (Section 29.3). Spliceosomes recognize signals in the nascent RNA that specify the splice sites. Introns nearly always begin with GU and end with an AG that is preceded by a pyrimidine-rich tract. This consensus sequence is part of the signal for splicing..  Alternative splicing Alternative splicing generates mRNAs that are templates for different forms of a protein: (A) a membrane-bound antibody on the surface of a lymphocyte and (B) its soluble counterpart, exported from the cell. The membrane-bound antibody is anchored to the plasma membrane by a helical segment (highlighted in yellow) that is encoded by its own exon. The flow of genetic information is from DNA to RNA to proteins, a scheme called the central dogma. DNA has noncoding regions Splicing to called introns interspersed remove between the coding introns sequences called exons  Structure of one chain of DNA Simple and elegant Double helix structure Other structures (Bacterial DNA, RNA) DNA replication Function Splicing Transcription Translation Splicing