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Download Before you begin this in-class project, you will need the following
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Before you begin this in-class project, you will need the following items which can be found at your local dollar store/craft store: Pipe cleaners Colored plastic beads Transcription and Translation Project http://learn.genetics.utah.edu/ Transcription is a process in which genes, or segments of DNA which encode for mRNA and ultimately a protein, are “turned on.” When this process of gene activation occurs, the segment of double-stranded DNA containing the gene is unwound and “opened.” A protein named RNA Polymerase II begins transcribing the DNA when it finds a start transcription sequence, or TATAA box. Transcribe the gene below using the bottom strand as the template strand. (A in DNA U in mRNA, C in DNA G in mRNA, G in DNA C in mRNA, T in DNA A in mRNA) Before the mRNA exits the nucleus, 3 post-transcriptional modifications occur: 1. Introns (intragenic sequences) are spliced out of the mRNA 2. On the 5’ end of the mRNA, a 5’-methyl-guanosine cap is added 3. On the 3’ end of the mRNA, a poly-A tail is added mRNAs are exported out of the nucleus and dock at the ribosomes that lie along the Rough Endoplasmic Reticulum (RER) and are freely floating in the cytoplasm. The two subunits of the ribosome clamp onto the end of the mRNA and begin scanning mRNA for a start translation site, or AUG site mRNA is translated into amino acids when read in groups of 3 nucleotides, or codons (see universal codon chart in your book or on-line). Stop sequences are also encoded by either an UGA, UAA, or an UAG. When a stop codon is encountered, the ribosome complex disassociates and the polypeptide sequence is complete. The polypeptide then is folded into a protein (Lodish, 2007). Nucleus DNA 5’ A T A T T C A T T A T G C C C A A A G G G T T T T A G C C 3’ Non-template 3’ T A T A A G T A A T A C G G G T T T C C C A A A A T C G G 5’ Template Transcription mRNA: 5’ ________________________________________________________3’ Translation Protein : Amino-_______________________________________________________________________-COOH Ribosomes mRNA Mutations are changes in the DNA sequence. Sometimes, the mutation leads to a change in the resultant polypeptide sequence and sometimes it doesn’t. Some common mutations are: a single nucleotide being converted into a wrong nucleotide, deletions of 1 or many nucleotides, or insertions of 1 or many nucleotides. For codon information, search for codon chart. DELETION: DNA T deleted ΔT 5’ A T A T T C A T T A T G C C C A A A G G G T T T A G C C 3’ Non-template 3’ T A T A A G T A A T A C G G G T T T C C C A A A T C G G 5’ Template mRNA: 5’ 3’ Protein: Amino- - COOH INSERTION: DNA T inserted 5’ A T A T T C A T T A T G C C C T A A A G G G T T T T A G C C 3’ Non-template 3’ T A T A A G T A A T A C G G G A T T T C C C A A A A T C G G 5’ Template mRNA: 5’ 3’ Protein: Amino- -COOH Below is a representation of the col1A gene the α-helical protein found in NORMAL COLLAGEN 1A protein. Transcribe and translate its sequence: DNA 5’ ATG GAA GGG AGT AAA GGC TAT GAC ACA GGT CGT TCG GGC GAT TAC GGG CGC TGA 3’ Non-temp. 3’ TAC CTT CCC TCA TTT CCG ATA CTG TGT CCA GCA AGC CCG CTA ATG CCC GCG ACT 5’ Template mRNA: 5’ Protein: Amino- 3’ -COOH Using colored beads and pipe cleaners, demonstrate the primary sequence of the COLLAGEN 1A sequence determined above. Use the legend below to determine which colored bead represents which amino acid. Then, based upon your understanding of ionic interactions (negatives and positives are attracted to each other), conform your primary sequence into its appropriate secondary alpha-helical structure on to your pipe cleaner. YELLOW BEADS = NON-POLAR, HYDROPHOBIC: Gly, Ala, Val, Leu, Ile, Pro, Phe, Tyr, Trp GREEN BEADS = POLAR, UNCHARGED: Met, Ser, Thr, Cys, Asp, Glu BLUE BEADS = POSITIVELY CHARGED: Lys, Arg, His RED BEADS = NEGATIVELY CHARGED: Asp, Glu Below is a representation of the col2A gene the α-helical protein found in NORMAL COLLAGEN 2A protein. Transcribe and translate its sequence and conform the resultant primary sequence on to your pipe cleaner: DNA 5’ ATG CGA GGG AGT GAG GGC ACG AAG GGA CAA GAG GGT CAC AGG GGG AGC GAA ATG 3’ 3’ TAC GC T CCC TCA CTC CCG TGC TTC CCT GTT CTC CCA GTG TCC CCC TCG CTT TAC 5’ Template mRNA: 5’ Protein: Amino- 3’ -COOH Osteogenesis imperfecta: caused by a loss in or mutations in collagen (genes: col1A, col2A) this disrupt collagen quaternary structure. It is often referred to as “brittle bone disease” as individuals with the condition suffer from bones that break very easily. Their bones may also be quite deformed (http://www.niams.nih.gov/Health_Info/Bone/Osteogenesis_Imperfecta). Below is a mutated col1A gene the mutated α-helical protein found in the mutated COLLAGEN 1A protein associated with some forms of osteogenesis imperfecta. Transcribe and translate its sequence and conform the resultant primary sequence on to your pipe cleaner: DNA 5’ ATG GAA GGG AGT AAC AGC TTG TCG TAT GAC ACA GGT CGT TCG GGC GAT TAC GGG CGC TGA 3’ Non-temp. 3’ TAC CTT CCC TCA ATA CTG TGT CCA GCA AGC CCG CTA ATG CCC GCG ACT 5’ Template mRNA: 5’ 3’ Protein: Amino- -COOH * Notice the pattern of the beads has been altered in the mutant. This disrupts the α-helical structure and impairs function of collagen! Normal Collagen Quaternary Structure Mutated Collagen Quaternary Structure resulting in Osteogenesis imperfecta (Figure used from http://www.cell.com/cms/attachment/579430/4340625/gr1.jpg) Critical Thinking Questions 1. What type of chemical bonds form in between amino acids in the primary structures of all proteins? 2. What type of chemical bonds form in between the negatively charged and positively charged amino acids in collagen? 3. When the quaternary structure of collagen forms, the triple α-helix, what type of weak interactions maintain its complex structure between the polar, hydrophilic amino acids between each protein chain? 4. Name and describe all the levels of protein structure that are ultimately affected by the mutation shown causing osteogenesis imperfecta? 5. Which of these types of chemical interactions are disrupted by heat? By the addition of salt? By the addition of hydrolases (enzymes that catalyze hydrolysis reactions)? 6. Besides bone, what other types of problems/system issues might someone with osteogenesis imperfecta experience?