DNA Structure & Replication Review Slides PDF

DNA Structure Central Dogma: DNA > RNA > Protein DNA Structure DNA is a nucleic acid composed of nucleotides 5-carbon sugar called deoxyribose Phosphate group (PO4) Attached to 5′ carbon of sugar Nitrogenous base Adenine, thymine, cytosine, guanine Free hydroxyl group (—OH)...

DNA Structure Central Dogma: DNA > RNA > Protein DNA Structure DNA is a nucleic acid composed of nucleotides 5-carbon sugar called deoxyribose Phosphate group (PO4) Attached to 5′ carbon of sugar Nitrogenous base Adenine, thymine, cytosine, guanine Free hydroxyl group (—OH) Attached at the 3′ carbon of sugar Complementary Base Pairing: A-T; GC Double helix formed by complementary strands 2 Central Dogma Information flows from DNA to RNA to Protein Replication DNA to DNA Transcription DNA to RNA Translation RNA to Proteins Occurs in different compartments of the cell Information Flow in the Cell Nucleus Replication DNA to DNA Transcription DNA to RNA Splicing Pre-mRNA to mRNA Rough Endoplasmic Reticulum Translation mRNA to Protein Protein folding Misfolded proteins degraded in the cytoplasm Golgi Apparatus Modifies and transports proteins DNA Replication or Synthesis Occurs at the S Phase of the Cell Cycle  DNA Replication Every time a cell divides, its entire genome must be duplicated both quickly and without mistakes. Watson and Crick postulated that DNA replication could be as simple as unzipping the helix and replacing the missing nucleotides. DNA Replication begins at discrete origins and then proceeds bidirectionally DNA replication in E. coli visualized by autoradiography of radioactive thymidine  DNA Polymerase DNA polymerase is the main enzyme in DNA replication. It catalyzes the joining of deoxyribonucleotide 5′- triphosphates (dNTPs). DNA Polymerase I First DNA polymerase was identified from E. coli in 1956 by Arthur Kornberg and colleagues using a combination of genetics and biochemical approaches. Both prokaryotic and eukaryotic cells contain several different DNA polymerases that play distinct roles in DNA replication and repair. In addition to a DNA polymerase, several other key proteins are required at the replication fork The enzymes involved in DNA replication act in a coordinated manner to synthesize both leading and lagging strands of DNA simultaneously. The leading and lagging polymerases are coordinated by their association with Clamp- loading protein (i.e., RFC). All DNA Polymerases share two fundamental properties 1. Synthesize DNA only in the 5′ to 3′ direction. 5 3 2. Add new doxynucleotide triphosphates (dNTPs) only to a primer strand that is hydrogen-bonded to a template; they cannot initiate DNA synthesis from free dNTPs.  If a primer is needed, how does DNA polymerase begin replication? RNA synthesis can initiate de novo. An enzyme called DNA Primase synthesizes short fragments of RNA that act as primers. These short fragments of RNA then serve as primers for synthesis of DNA.  The Fidelity of DNA Replication DNA polymerase helps select the correct bases for insertion. Binding of correctly matched dNTPs induces conformational changes in DNA polymerase that lead to the incorporation of the nucleotide. Together with base-pairing, this reduces the error rate to about 1 in 105 per nucleotide.  Synthesis of leading and lagging strands The solution is to have one strand of DNA synthesized in the 5′ to 3′ direction and in a continuous manner (the leading strand). The other (lagging strand) is formed from short, discontinuous pieces of DNA that are also synthesized 5’ to 3’, but in an overall forward direction. These small pieces (Okazaki fragments) are joined by DNA ligase. Linear Chromosomes of Eukaryotes have an End Replication Problem In the next cell generation, this template will be missing DNA (lagging strand)  Telomeres and Telomerase The terminal sequences of linear DNA molecules (telomeres) consist of tandem repeats of simple- sequence DNA. The terminal sequences are maintained by telomerase, which catalyzes synthesis of telomeres in the absence of a DNA template. Telomerase is a reverse transcriptase, a class of DNA polymerases that use an RNA template instead of a DNA template. Telomerase carries its own template RNA, which is complementary to the telomere repeat sequences. Telomerase (after many rounds of telomerase extension)  Telomerase Removal of the RNA primer leaves an overhanging 3′ end, which can form loops at the ends of eukaryotic chromosomes.  Transcription: DNA to RNA Prokaryotes vs Eukaryotes Bacteria and eukaryotes handle their mRNA transcripts differently Promoter ! Longer mRNA transcripts have more ribosomes ! Recall that bacteria have no nucleus; Gene ! therefore transcription and translation take place in the same compartment. ! Bacterial mRNAs are used immediately RNA polymerases for protein synthesis—while still being that began transcription eariler transcribed. ! Bacterial mRNA have a very short half life, typically just a few minutes. Bacteria and eukaryotes handle their mRNA transcripts differently ! In eukaryotes, transcription and processing take place in the nucleus, but translation takes place in the cytoplasm. ! Transcription and translation are physically and temporally separated in eukaryotes. ! Therefore, the mRNA must be exported to the cytoplasm via pores in the nuclear membrane.  Regulation of Transcription Regulation of gene expression allows a bacteria cell to adapt to environmental changes, such as food sources. Most transcriptional regulation in bacteria operates at the initiation step. In addition to the promoter, nearly all genes (whether bacterial or eukaryotic) have regulatory DNA sequences. In bacteria, this sequence is commonly called an operator.  Regulation of Tryptophan Operon Several proteins are needed to synthesize tryptophan; a cell will only make these proteins if needed. These genes are expressed as part of a single mRNA molecule and are therefore controlled by one promoter—clusters like these are called operons. The tryptophan operon is controlled by a strong promoter—it will bind RNA polymerase and transcribe the operon unless it is repressed.  Regulation of Tryptophan Operon The tryptophan operon is switched off by a repressor protein, the tryptophan repressor. In turn, the tryptophan repressor is responsive to tryptophan levels.  Lactose Metabolism Three enzymes are involved: "-galactosidase (z) cleaves lactose into glucose and galactose. Lactose permease (y) transports lactose into the cell. Transacetylase (a) inactivates toxic thiogalactosides that are transported into the cell along with lactose. Genes encoding these enzymes are expressed as a single unit: the Lac Operon.  The combined action of activators and repressors control the lac operon Studies of gene regulation by Jacob and Monod in the 1950 s elucidated the transcriptional regulation of the enzymes involved in lactose metabolism. These enzymes are only expressed when glucose is absent and lactose is present.  Negative control of the lac operon Two loci control transcription: – o (operator), adjacent to transcription initiation site – i (i gene, not in the operon), encodes a protein that binds to the operator. In the presence of lactose, the repressor is inactivated  Positive control of the lac operon The presence of glucose (a preferred energy source) represses expression of the lac operon, even if lactose is also present. This is mediated by a positive control system: If glucose decreases, levels of cAMP increase. cAMP binds to the regulatory protein catabolite activator protein (CAP). This stimulates CAP to binds to its target DNA sequence upstream of the lac operon. CAP facilitates binding of RNA polymerase to the promoter. Eukaryotic Transcriptional Regulation More complex than prokaryotes Eukaryotes have DNA organized into chromatin. Complicates protein-DNA interaction Eukaryotic transcription occurs in nucleus while translation occurs in cytoplasm. Amount of DNA involved in regulating eukaryotic genes much larger. 27 Eukaryotic chromatin structure DNA wound around histone proteins & non- histone regulatory proteins to form nucleosomes Nucleosomes and histones complicates the process of transcription (that is restricts access of the transcription machinery to the DNA). Chromatin structure is selectively modulated to allow transcription. 28 Promoters and enhancers Promoters DNA binding sites for general transcription factors Mediate the binding of RNA polymerase II to the promoter. Enhancers DNA binding sites for specific transcription factors Act over long distances by bending DNA to form loop to position enhancer closer to promoter. Gene regulation can occur at great distances Enhancers, like promoters, function by binding transcription factors that then regulate RNA polymerase. DNA looping allows a transcription factor bound to a distant enhancer to interact with proteins associated with the RNA polymerase/Mediator complex at the promoter. Post-transcriptional Regulation Eukaryotic genes contain introns and exons (b): Courtesy of Dr. Bert O’Malley, Baylor College of Medicine Access the text alternative for slide images. 32 Post-transcriptional regulation: pre-mRNA splicing Pre-mRNA splicing removes introns and joins exons to produce mature mRNA. snRNPs (small nuclear ribonucleoproteins) recognize intron-exon boundaries Spliceosome A complex of snRNPs and proteins that catalyze splicing. Introns are excised and exons are ligated to form the mature mRNA Alternative splicing A process where a single gene produces multiple mRNA variants by including or excluding different exons. 33 Alternative splicing Definition: A process where a single gene produces multiple mRNA variants by including or excluding different exons. Tissue-Specific Splicing: Different tissues can produce distinct protein forms from the same gene. Example: The calcitonin gene produces: Calcitonin in the thyroid (regulates calcium). Calcitonin gene-related peptide (CGRP) in the hypothalamus (involved in pain signaling). 34  Translation: mRNA to protein Cotranslational targeting of secretory proteins to the ER (step 5) The signal sequence is cleaved by signal peptidase and released into the ER lumen.  Elongation – Part 1 Ribosomes have 3 binding sites: P (peptidyl), A (aminoacyl), and E (exit) sites (large subunit). The P site contains the growing polypeptide and is still held by the tRNA (the peptidyl tRNA) bound to its codon, or if protein synthesis is just beginning, a methionine (methionyl tRNA). In the first step, an aminoacyl tRNA binds to the A site by pairing with the next codon of the mRNA. An elongation factor (EF-Tu in prokaryotes, eEF1! in eukaryotes) complexed to GTP brings the aminoacyl tRNA to the ribosome. Insertion of the correct aminoacyl tRNA at the A site triggers a conformational change that induces hydrolysis of GTP/eEF1! and release of the elongation factor.  Elongation – Part 2 Then the peptide bond is formed: the carboxyl end of the polypeptide is uncoupled from the tRNA in the P site and joined to the free amino group of the amino acid linked to the tRNA in the A site. This is mediated by the rRNA of the large subunit; 23S in prokaryotes, 28S in eukaryotes. In the process of peptide bond formation, the polypeptide is transferred to the A site and the remaining tRNA in the P site is uncharged.  Elongation – Part 3 Next is translocation: the ribosome moves three nucleotides along the mRNA, positioning the next codon in an empty A site. This step translocates the peptidyl tRNA from A to P, and the uncharged tRNA from P to E. A new aminoacyl tRNA binds to the A site (not shown in this cartoon) and induces release of the uncharged tRNA from the E site. Translocation requires another elongation factor (EF-G in prokaryotes, eEF2 in eukaryotes) and is coupled to GTP hydrolysis. With the growing polypeptide linked to the tRNA in the P-site, the elongation cycle can begin again to add the next amino acid. Post-transcriptional Regulation  Lin-4 is a microRNA (miRNA) that Represses the Translation of Lin-14 Drosha Processes Pri-miRNA into dsRNA Hairpins Dicer Creates Functional Form of miRNA RISC = RNA-Induced Silencing Complex Other miRNAs can regulate the translation of whole groups of mRNAs Tissue specific expression of As many as 1000 miRNAs miRNA in zebrafish embryos are encoded in mammals; Nervous system each can target up to 100 different mRNAs. At least one-third of protein-coding genes may Skeletal muscle be regulated by miRNAs. miRNAs are important in embryonic development, Liver and may contribute to heart disease and cancer. Protein folding and quality control choices for a newly made protein If quality control fails, a possible outcome is the formation of protein aggregates.  Defects in protein folding are responsible for many diseases Alzheimer’s disease is associated with two types of lesions in brain tissue caused by accumulation of misfolded proteins: Plaques of amyloid-! protein, which is a cleavage fragment that misfolds and then inappropriately accumulates and aggregates. Tangles form that are insoluble aggregates of protein tau that have undergone abnormal phosphorylation.