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33 Cards in this Set
- Front
- Back
Characteristics of the Genetic Code
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1. Linear code of ribonucleotide letters
2. "words" are triplets of ribonucleotide i.e. codons 3. Code is unambiguous codon specifiies one aa 4. Code is degenerate 1 or more codons/ aa 5. Start and stop codons initiate and terminate translation 6. Translation without interruption (comma-less) 7. Non-overlapping code 8. Code is nearly universal |
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How is genetic information encoded?
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mRNA
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mRNA
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unstable intermediate between DNA and the ribosomes that transfer the genetic information from DNA to proteins
mRNA is relatively unstable, especially when compared to DNA |
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How are 20 aa's specified from 4 nucleotides?
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Brenner proposed that the genetic code existed as a triplet which would allow for 64 combinations of the 4 letters
Crick and colleagues found the frameshifts of 1 or 2 nucleotides caused mutations Gain or loss of 3 nucleotides did not cause mutation. |
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Deciphering the Genetic Code: Nirenberg
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Synthesized mRNAs using the enzyme Polynucleotide Phosphorylase
Used these artificial messages in an in vitro protein synthesis system to make polypeptide sequences. Does not require DNA template- adds ribonucleotides randomly Ribonucleotide that is added is dependent on the concentration of that ribonucleotide relative to the other three. |
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Homopolymers
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Polymers of single nucleotides
e.g. polyU, polyA, polyG Use homopolymers in in vitro translation and determine which amino acid was added to the newly synthesized protein Used a mixture of the 20 amino acids, but only one was radioactively labeled so that it could be detected. |
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RNA Heteropolymers (Copolymers)
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Add two or more different ribonucleotides to form the artificial mRNA.
Based on the ratio of ribonucleotides added, the composition of the codons can be predicted: Codon composition that is predicted to occur most frequently would be predicted to specify the amino acid that is found in the greatest amount in the peptide. Allows the composition of the triplet codons to be deduced (although not the exact sequence of the codon) |
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Triplet Binding Experiments to Determine Sequence of Codons
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Synthesized small tri-ribonucleotides
Ribosomes would bind these and cognate tRNA Radioactively label single amino acids Identify which amino-acid bound tRNA associated with the ribosome/tri-ribonucleotide complex i.e. which labeled amino acid would be retained in the ribosome complex. |
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Sequencing MS2 phage
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the DNA/RNA sequence and amino acid sequence of the encoded proteins were shown to be "co-linear"
5' end of the gene = NH terminal of the protein |
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Universality of the Genetic Code
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MS2 phage
Cell free bacterial systems used to translate eukaryotic genes Recombinant DNA molecules expressed correctly in different organisms. |
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The Genetic Code
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Dictionary of 61 triplets (codons) for 20 amino acids
3 codons specify terminatino: UAA, UAG, and UGA First two nucleotides in the codon more critical for attracting the correct tRNA to the mRNA. 3rd position = wobble position |
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Stop Codons
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UAA, UAG, UGA
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Wobble position
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3rd position; has relaxed base pairing rules
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Translation start codon
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AUG (prokaryotes & eukaryotes)
rarely GUG in prokaryotes Specifies N-formylmethionine (fmet) formyl group removed after the protein is synthesized. |
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Transcription
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Synthesis of RNA molecules form DNA template
RNA molecule is complementary to one strand of the DNA template Triplet codons in the mRNA strand is complementary to an anticodon region of a specific tRNA molecule mRNA transfers genetic information from DNA to protein. |
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E. coli polymerase
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B a B' subunits provide the catalytic activity
a subunits provide scaffolding function and interact with other proteins o factor provides a regulatory function All RNA polymerases synthesize RNA in a 5'-3' direction |
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O factor
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Different o factors provide specificity for binding of RNA pol to different promoters
o factor is not part of the holoenzyme. |
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Bacterial Promoter Elements
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-35 and -10
- 35 : TTGACA - 10: TATAAT (pribnow box = -10 region) |
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3 stages of Transcription
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Initiation, Elongation, Termination
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Initiation
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Polymerase binds to the promoter sequence in duplex DNA (closed complex)
Polymerase melts duplex DNA near transcription start site, forming a transcription bubble (operon complex) Polymerase catalyzes phosphodiester linkage of two initial rNTPs. |
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Elongation
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Polymerase advances 3'-5' down template strand, melting duplex DNA and adding rNTPs to growing RNA.
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Termination
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At transcription stop site, polymerase releases completed RNA and dissociates from DNA.
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Eukaryotic Transcription
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Transcription and Translation occur in separate compartments: no coupling of transcription and translation
More DNA sequences and proteins involved in activating transcription Eukaryotic mRNAs undergo post-transcriptional processing. |
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Post-transcriptional processing
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addition of 5' cap structure and 3' poly-A tail
Removal of introns and splicing exons |
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Introns
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intervening sequences (split genes)
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3 forms of RNA polymerase in Eukaryotic Cells
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I, II, and III.
I: rRNA II: mRNA III: tRNA |
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Initiation of Eukaryotic Transcription
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cis-acting elements- DNA sequencing elements: serve as binding sites for trans-acting factors
e.g. TATA box, CAAT box, enhancers |
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trans-acting factors
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transcription factors
bind cis-acting elements Function to recruit RNA polymerase and promote transcription initiation |
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General (Basal) Transcription Factors
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TFIID- complex that contains TATA box-binding protein (TBP)
Associate with the promoter at the TATA box and recruit RNA Pol II to form the Pre-initiation complex. |
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Transcriptional Activators and Repressors
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Bind to enhancer elements to induce high levels of transcription or to repressor elements to repress transcription.
SP-1, NF-kB, CREB |
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Steps in Transcription Initiation in Eukaryotes
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1. TFIID binds to the TATA box
2. Recruites other general factors and the RNA Pol II holoenzyme to form the PIC 3. Helicase activity of TFIIH unwinds the DNA at the promoter and allows RNA Pol II to initiate transcription 4. Once elongation begins, most of the general factors dissociate. |
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Posttranscriptional Processing of RNA in Eukaryotes
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Pre-mRNA= heterogeneous RNA or RNA that has not been processed
RNA processing occurs in nucleus As the new strand of RNA is being transcribed, a 5' cap is added RNA processing at the 3' end of the transcript: poly-adenylation. Splicing. |
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3' Poly-adenylation
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a portion of the 3' end is cleaved and Poly-A Polymerase adds about 200 adenosine residues to the 3' end.
Poly A tail functions primarily in mRNA stability. |