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PHYSIOLOGY. Signal Transduction and Protein Synthesis. DNA. DNA Deoxyribonucleic Acid Twisted ladder or double helix Nucleotides Composed of alternating sugar (Deoxyribose) and phosphate molecules and Nitrogen bases Purines = adenine and guanine Pyrimidines = thymine cytosine. DNA.

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  1. PHYSIOLOGY Signal Transduction and Protein Synthesis

  2. DNA • DNA • Deoxyribonucleic Acid • Twisted ladder or double helix • Nucleotides • Composed of alternating sugar (Deoxyribose) and phosphate molecules and • Nitrogen bases • Purines = adenine and guanine • Pyrimidines = thymine cytosine

  3. DNA • Purines bond with Pyrimidines • Complementary base pairs • Adenine with Thymine • Guanine with Cytosine

  4. DNA • Purines bond with Pyrimidines • Complementary base pairs • Adenine with Thymine • Guanine with Cytosine • Nucleoside • Sugar bonding with a base • Nucleotide • Adding a phosphate to a nucleoside • Phosphates attach to the 5’ carbon of the sugar

  5. Orientation of DNA The carbon atoms on the sugar ring are numbered for reference. The 5’ and 3’ hydroxyl groups (highlighted on the left) are used to attach phosphate groups. • The directionality of a DNA strand is due to the orientation of the phosphate-sugar backbone.

  6. DNA is a double helix P A T P 5’ 3’ P C G P P P G C A P P P T P G C P P C G 3’ 5’ P P A sugar and phosphate “backbone” connects nucleotides in a chain. DNA has directionality. Two nucleotide chains together wind into a helix. Hydrogen bonds between paired bases hold the two DNA strands together. DNA strands are antiparallel.

  7. DNA • A chromosome • 23 pair = diploid • 23 = haploid; sex cells • Duplicating DNA structure tightly packed around histone proteins to form a nucleosome.

  8. DNA • A gene • A series of bases that occupy a specific location (locus) on a chromosome • The code of a single protein or polypeptide • Genetic Alphabet • Triplet = Three nucleotides on DNA with their corresponding base pairs making up the code of a single amino acid • Codon = Three successive nucleotides on RNA with their corresponding base pairs making up the code of a single amino acid • 20 amino acids • A series of amino acids makes up a protein

  9. DNA • Consists of 3 billion base pairs • Codes for about 50 to 100,000 genes • Genes may exist in alternate forms = alleles • One allele from mom and one allele from dad • Nucleotide changes or mutations may occur in a gene • Sickle cell anemia • In a healthy population, a gene may exist in multiple alleles • Genetic Polymorphism = Multiple different forms at a gene locus in a population • Basis for DNA typing using MHC

  10. Terminology • Allele • An alternate form of a gene • Locus • Location of a gene on a chromosome • Gene • Genetic code or “blueprint” for the cell to build one particular protein

  11. http://www.youtube.com/watch?v=983lhh20rGY

  12. http://www.youtube.com/user/ndsuvirtualcell?blend=10&ob=5 • http://www.youtube.com/watch?v=YjWuVrzvZYA • http://www.youtube.com/watch?v=FVuAwBGw_pQ&feature=related • http://www.youtube.com/watch?v=5bLEDd-PSTQ&feature=related

  13. http://www.youtube.com/watch?v=NJxobgkPEAo

  14. Two types of nucleic acids • DNA • Usually double-stranded • Has thymine as a base • Deoxyribose as the sugar • Carries RNA-encoding information • Not catalytic • RNA • Usually single-stranded • Has uracil as a base • Ribose as the sugar • Carries protein-encoding information • Can be catalytic

  15. Protein Synthesis Proteins are necessary for cell functions Protein synthesis is under nuclear direction  DNA specifies Proteins DNA mRNA Protein ? ?

  16. 1 start codon 3 stop codon 60 other codons for 19 aa Redundancy of Genetic Code (p 115) A combination of three bases forms a codon

  17. RNA • Definitions • Exon • Amino acid specifying informational sequences in the genes of higher organisms • Intron • Noncoding segments or portions of DNA that ranges from 60 to 100,000 nucleotides long

  18. Transcription DNA is transcribed into complementary mRNA by RNA Polymerase + nucleotides + Mg2+ + ATP Gene = elementary unit of inheritance Compare to Fig. 4-33

  19. http://vcell.ndsu.edu/animations/

  20. Transcription • First steps in protein synthesis that occurs completely within the nucleus • DNA is used as a template to create a small single strand of mRNA that can leave through the nuclear pore. • The enzyme RNA polymerase plus magnesium or manganese ions along with ATP are needed in this process.

  21. Transcription DNA is used as a template for creation of RNA using the enzyme RNA polymerase. DNA 5’ G T C A T T C G G 3’ 3’ C A G T A A G C C 5’

  22. 3’ G U C A U U C G G 5’ RNA Transcription The new RNA molecule is formed by incorporating nucleotides that are complementary to the template strand. DNA DNA coding strand 5’ G T C A T T C G G 3’ 3’ C A G T A A G C C 5’ • DNA template strand

  23. Transcription • Promoter • Sequence on DNA where the RNA polymerase attaches to begin transcription • A region at the beginning of a gene that must be activated before transcription can begin. • This region is not transcribed into mRNA

  24. Transcription • Transcription Factors • Binds to DNA and activates the promoter • Tells the RNA polymerase where to bind to the DNA • RNA polymerase moves along the DNA molecule and “unwinds” the double strand by breaking hydrogen bonds between base pairs • Sense strand • Guides RNA polymerase in RNA synthesis • Antisense strand • Sits idly by and is not transcribed

  25. Transcription • Each base of the DNA sense strand pairs with a complementary mRNA base • AGTAC on DNA • UCAUG on mRNA • Uracil is substituted for Thymine • Ribose sugar is used as the backbone of mRNA instead of Deoxyribose sugar

  26. TATA binding protein Gene sequence to be transcribed Promoter DNA GG TATA CCC TATA box Transcription begins Transcription factor Initiation of transcription Transcription begins at the 5’ end of the gene in a region called the promoter. The promoter recruits TATA protein, a DNA binding protein, which in turn recruits other proteins.

  27. mRNA processing • Alternative splicing occurs • Enzymes clip segments out of the middle or off the ends of mRNA strands • Introns • mRNA segments are spliced back together by the spliceozyme enzyme • Exons • The processes mRNA leaves through the nuclear pore and attaches to a ribosome

  28. mRNA • Contains the coded information for the amino acid sequence of a protein • 3 main parts: • 5' leader sequence - important for the start of protein synthesis. • Coding Sequence - the sequence that codes for the amino acid. • 3’ trailer sequence - poly A tail.

  29. Messenger RNA undergoes three (or four) post-transcriptional modifications • 1. Capping of 5’ end 2. Additional of poly A tail to 3’ end 3. Removal of introns 4. Editing of RNA (rarely) EUKARYOTES ONLY!!!!!!!!!!!!!!!!

  30. 5’ capping. Involves the addition of a guanine (usually 7-methyl-guanosine) to the terminal 5’ nucleotide. • The enzyme that completes this process is called a capping enzyme. • The 5’ cap is required for the ribosome to bind to the mRNA as the initial step of translation.

  31. Addition of a 3’poly A tail. • This poly(A) tail is usually about 50 - 250 bps of adenine in length. • There is no DNA template for this tail? • Poly A tails are found on most mRNA molecules but not all (ex. histones mRNA have no poly A tail). • In general, a eukaryotic mRNA molecule is longer than the required transcript. The enzyme RNA endonuclease cleaves the molecule at the poly(A) addition site to generate a 3’ OH end. • The poly A tail is important for determining the stability of the mRNA molecule so the mRNA doesn’t degrade.

  32. Translation • Translation begins when mRNA binds to a ribosome in the cytoplasm of the cell.

  33. Translation mRNA is translated into string of aa (= polypeptide) 2 important components ?? mRNA + ribosomes + tRNA meet in cytoplasm Anticodon pairs with mRNA codon  aa determined Amino acids are linked via peptide bond.

  34. The Genetic Code • The code has start and stop signals. AUG (methionine) is the common start codon Methionine can also be used WITHIN a polypeptide GUG may also be used as a start codon. • There are three stop codons. UAG UAA UGA All three are chain termination codons.

  35. Ribosomal RNA • Large and small subunits • Binding sites • One for mRNA • Three for tRNA • P site = Peptidyl-tRNA site • A site = Aminoacyl-tRNA site • E site = Exit site

  36. Transfer RNA • The correct amino acid is added to the growing polypeptide only if: • 1 - The appropriate amino acid is added to the tRNA by aminoacyl-tRNA synthetases. • 2 – Complementary binding occurs between the codon of the mRNA and the anticodon of the tRNA.

  37. Translation (An Overview) • Translation is defined as protein synthesis. • Occurs on ribosomes, where the genetic information is translated from the mRNA to a protein. • mRNA is translated in the 5’ to 3’ direction. • Amino acids are brought to the ribosome bound to a specific tRNA molecule. • The mRNA and tRNA are responsible for the correct recognition of each amino acid in the growing polypeptide

  38. Initiation • A small ribosomal subunit binds to both mRNA at the 5’ cap along with a specific initiator tRNA • The initiator tRNA carries methionine • tRNA’s anticodon binds with the codon on mRNA • The large ribosomal subunit attaches to form the translation initiation complex. • The initiation complex is held together by proteins called initiation factors

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