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The “Central Dogma” Overview Of DNA How does DNA control the cell?. Transcription. Translation. DNA. mRNA. proteins. Enzymes Structure Movement Hormones Gas exchange Amino Acid Storage. Replication. So how does DNA “control” the cell?. Transcription. Translation. DNA. mRNA.
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The “Central Dogma”Overview Of DNAHow does DNA control the cell? Transcription Translation DNA mRNA proteins Enzymes Structure Movement Hormones Gas exchange Amino Acid Storage Replication
So how does DNA “control” the cell? Transcription Translation DNA mRNA proteins Enzymes Structure Movement Hormones Gas exchange Amino Acid Storage Replication
Erwin Chargaff • A = 30.9% • T = 29.4% • G = 19.9% • C = 19.8%
From DNA to Chromosome chromosome nucleus • A strand of human DNA is about 3 m long… • How does it fit into all our cells?? • Supercoiling cell Proteins that DNA wraps around histones Base pairs DNA
Details of DNA Structure • Nucleotides are the monomers of nucleic acids • 5 carbon sugar • Ribose • Deoxyribose • Nitrogen Base • Adenine • Thymine • Cytosine • Guanine • Uracil • Phosphate 5’ Carbon 5’ 4’ 1’ 2’ 3’ 3’ Hydroxyl
Details of DNA Structure 5’ Carbon 3’ Hydroxyl • What do you notice about the 5’ and 3’ ends of the two strands? • They’re ANTIPARALLEL!! • Why? For the nucleotide bases to line up 3’ Hydroxyl 5’ Carbon
Details of DNA Structure 5’ Carbon 3’ Hydroxyl • What holds the nucleotides together? 3’ Hydroxyl 5’ Carbon
Details of DNA Structure • Nucleotide Bases: Purines and Pyrimidines • PURINES • “Aggies are Pure” – A and G are Purines which have 2 rings • PYRIMIDINES • “TCU Cheerleaders build Pyramids” – T and C are Pyrimidines have one ring
Key Questions • How do 3 m of DNA fit into each of our cells? (be specific!) • Why is a DNA molecule considered to have direction and to be “anti-parallel”? • What type of bonds form the sugar-phosphate backbone? • What type of bonds hold the nucleotide bases together? • From what you know about the bonding between base pairs, which pairs (A-T or G-C) do you think have more breaks and mistakes and why?
Question: • How does the structure of DNA ensure the daughter strands will be identical to the parent strand?
DNA Replication Parent strand Origin of replication Daughter strand Bubble Replication fork 2 new strands
DNA Enzymes • Helicase • Breaks hydrogen bonds to unwind DNA • DNA Polymerase III • Adds nucleotides ONLY to the 3’ end • Nucleoside PPP links to sugar-P backbone • Losing 2 Ps provides energy for bonding
Problem: Nucleotides can only be added to the 3’ end by DNA Polymerase… • Solution: Okazaki • Leading and Lagging Strands • Leading Strand • Continuous synthesis • Lagging Strand • Okazaki fragments • Joined by ligase
3’ Remember: DNA polymerase can only add nucleotides to the 3’ end, so DNA gets built in the 5’ 3’ direction! 5’ Parental DNA 5’ Okazaki fragments 3’ DNA polymerase 3’ Ligase Leading and lagging have the same origin of replication, but since DNA polymerase can only add on the 3’ end, the lagging strand has to start backwards and make little pieces to link together 5’ Leading strand One piece of 5’ 3’ Many little pieces of 5’ 3’ linked together later Lagging strand
Test your understanding…On some paper, write A – H and decide whether each letter represents the 3’ or 5’ end of DNA. Then, label the sections (A-B, C-D, etc) as “leading” or “lagging” A-B: Leading C-D: Lagging B C A D 5’ 3’ 3’ 5’ 3’ 5’ E H 3’ 5’ G F F-E: Leading H-G: Lagging
Priming DNA Synthesis • DNA polymerase can only extend an existing DNA molecule; it cannot start a new one • Short RNA primer is built first on parent DNA by primase • RNA primer later removed by DNA polymerase I
Priming DNA Synthesis • Closer look… Primase builds the RNA primer Replaces RNA nucleotides with DNA Primase DNA polymerase
Putting it all together! • http://www.johnkyrk.com/DNAreplication.html
Editing and Proofreading DNA Why do we not always get cancer? DNA can repair itself!!! • Since DNA polymerase III does 1,000 base pairs/second, it makes a lot of errors • DNA Polymerase I (only 20 bp/sec) excises mismatched bases, repairs the DNA, and removes the primer • DNA polymerase I reduces error from 1 in 10,000 bp to 1 in 100 million bp!!
Problems at the end… • Ends of chromosomes are “eroded” with each replication (don’t get fully copied) • Telomeres are expendable, non-coding sequences at the ends of the DNA strand • short sequence of bases repeated 1000s of times • TTAGGG in humans
Telomeres and Aging telomere • In the absence of telomerase, the telomere will become shorter after each cell division. When it reaches a certain length, the cell may cease to divide and die. telomerase Extended telomere
Putting it ALL together • Summarize the roles of the key enzymes • Label the diagram showing the steps of DNA replication • DNA Structure – Questions and Practice
Summary of Replication Enzymes Unzips DNA (breaks H-bonds between nucleotides) Builds RNA primer in leading strand and Okazaki fragments Adds DNA nucleotides (20 bp/s); replaces RNA primer with DNA; repairs errors in DNA Adds DNA nucleotides (1,000 bp/s) Joins Okazaki fragments (using phosphate groups)
In the diagram below, label the key enzymes and structures in DNA replication. Be sure to label 3’ and 5’ ends, too!
Protein Synthesis • How does DNA control the structure and function of the cell? it makes proteins! • Structure: collagen, elastin, keratin • Enzymes: catalase, amylase, sucrase, etc • Hormones: insulin, glucagon, etc • Amino acid storage: albumin, ovalbumin, etc
The “Central Dogma”Overview Of DNA Transcription Translation DNA mRNA proteins Enzymes Structure Movement Hormones Gas exchange Amino Acid Storage Replication
Protein Synthesis! • Transcription • http://www.johnkyrk.com/DNAtranscription.html • Translation • http://www.johnkyrk.com/DNAtranslation.html
Notas – From Gene to Protein Metabolism teaches us about genes • Metabolic defects caused by non-functional enzyme • Studying metabolic diseases suggested that genes specified proteins • PKY • Alkaptonuria (black urine) • Genes dictate the phenotype
1 gene – 1 enzyme hypothesis • Beadle and Tatum – 1941
1 gene – 1 enzyme hypothesis • Beadle and Tatum – 1941 • Compared different nutritional mutants of bread mold, Neurospora • Created mutations by X-ray treatments X-rays break DNA) • Wild type grows on “minimal” media (sugar) • Mutants require different amino acids because each mutant lacks a certain enzyme needed to produce a certain amino acid • Conclusion: Broken gene = non-functional enzyme • Problems with: • One gene – one enzyme • not all proteins are enzymes, and they’re coded by genes too • One gene – one protein • many proteins consist of several polypeptide, and each polypeptide has it’s own gene • One gene – one polypeptide?
Defining a gene… • “Defining a gene is problematic because small genes can be difficult to detect, one gene can code for several protein products, some genes code only for RNA, two genes can overlap, and there are many other complications.” – Elizabeth Pennisi, Science 2003 • How would YOU define a gene in your own words?
The “Central Dogma” Transcription Translation DNA mRNA Protein Reverse Transcription Replication
From nucleus to cytoplasm… • Where are the genes? in DNA on chromosomes in the nucleus • Where are proteins synthesized? on ribosomes (free or on the ER) in the cytoplasm • How does the information get from the nucleus to the cytoplasm? mRNA is made in the nucleus and can travel into the cytoplasm to the ribosomes
deoxyribose ribose A-T, C-G T-A, A-U, C-G Double Single
Transcription Basics • Initiation • RNA polymerase binds to promoter sequence on DNA • where to start reading = Promoter (initiation site) • which strand to read = template strand • direction on DNA = reads 3’5 builds 5’ 3’ • Elongation • RNA polymerase unwinds DNA ~20 bp at a time • Reads DNA 3’ 5’ • Builds RNA 5’ 3’ • No proofreading, about 1 error/105 bases • Many copies, short life, no problem • Termination • RNA polymerase stops at termination sequence • mRNA leaves nucleus through pores
RNA Processing or Editing • 5’ cap • protection • targets mRNA for ribosome • Poly-A tail • protection • leads mRNA out of nucleus • Spliceosome • composed of snRNPs (small nuclear ribonucleoproteins) • introns – intervening, interrupting = removed by spliceosome • exons – expressed
Putting it Together – Transcription to Translation • How does mRNA code for proteins? • How can you code for 20 aa with only 4 nucleotide bases (A, U, G, C)? • How can an alphabet of 4 letters (nucleotides) translate into an alphabet of 20 letters (aa)?!
Breaking the code • Nirenberg and Matthaei • Determined 1st codon – amino acid match • UUU coded for phenylalanine • Created artificial poly(U) mRNA • Added mRNA to test tube of ribosomes and nucleotides • mRNA synthesized a single amino acid polypeptide chain: phe-phe-phe-phe-phe-phe