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Student questions of the day…. 14 3’ lag gin Q1: Do both mitochondrial and nuclear DNA replicate once per cell cycle? A1: Good question. Nuclear DNA is replicated once per mitotic cell division cycle, mitochondrial DNA is replicated throughout the cell cycle. Discontinous DNA synthesis gs ng adi tra nd d ran st le 5’ 3’ lag gin g Q2: What determines where continuous or discontinuous DNA replication will take place? A2: This is a consequence of the “3’ end addition” addition” rule. le ng adi str an d d n a Primer removal by RNAase, DNA fragments act as primers to “fill in” gaps and ligase joins fragments str See Purves 11.6 5’ H. Sive MIT 2007 7.013 3.2.07 1. DNA repair Molecular Biology II (DNA repair RNA synthesis) 16 5’ 3’ 3’ double-stranded DNA 5’ GATTACA CTAATGT strand-separation (one strand shown) template 3’ GATTACA 3’ GATTACA CGAATGT Outcome of incorrect replication 3’ incorrect replication round 1 strand-separation replication round 2 GCTTACA GATTACA 3’ 3’ CGAATGT CTAATGT 3’ 3’ mutant DNA molecule wild type DNA molecule Protective suits for people with Xeroderma, a DNA repair disorder H. Sive MIT 2007 17 5’ 3’ GATTACA CTAATGT See Purves 11.19 strand-separation template GATTACA 3’ TAATGT double-stranded DNA 5’ Proofreading 3’ replication of strand-separation Mismatch repair Me 3’ template 3’ A Me Me G TTACA C AATGT G 3’ 3’ double-stranded DNA 5’ GATTACA CTAATGT Me Me Me 3’ 5’ removed by 3’ to 5’ exonuclease “proofreading site” on DNAPol GATTACA 3’ ATAATGT incorrect base!!! GATTACA CTAATGT correct base!! Me Me Me 5’ 3’ See Purves 11.19 complementary strand requires “base pairing site” on DNAPol 5’ 18 GATTACA C AATGT incorrect base incorporated, makes a “bubble”, without H bonds 3’ incorrect removed correct base filled in Me Me Me DNA polymerase tries again H. Sive MIT 2007 3’ H. Sive MIT 2007 GATTACA CTAATGT 3’ How do repair enzymes know which strand is correct? Parental “A/C”s are methylated. BASE Quiz!!! 2’ OH on sugar identifies ribose and a ribonucleotide This is a ribonucleotide. Identify the chemical group that tells you this? GUCUCAGAG What type of polynucleotide is this? How do you know? RNA RNA Uracil instead of Thymine DNA 2. Transcription Principles 1 2 Transcription: basic idea Transcription from specific strand/position nucleus 5’ gene (dsDNA) 5’ strands 5’ separate, 5’ one copied (transcribed) into RNA gene (dsDNA) 5’ RNA is final product or translated into protein cytoplasm RNA (transcript) H. Sive MIT 2007 non-template strand 3’ 3’ template strand 5’ 3’ 5’ template is copied 5’ RNA released 5’ 5’ 3’ 5’ start site 5’ See Purves 12.4 3 2. Transcription Questions Transcription: the simplified story 3’ stop site 3’ H. Sive MIT 2007 4 5 Question 1… ~109 base pairs, 1m DNA per nucleus Question 2… Which strand is template? (also defines transcription direction) ~5% DNA = genes How does RNA polymerase find genes? H. Sive MIT 2007 6 Reminder!! Complementary DNA strands are not transcribed into the same RNA top strand as template 5’ GATTACA 3’ 3’ CUAAUGU 5’ 5’ UGUAAUC 3’ 5’ GATTACA 3’ 3’ CTAATGT 5’ bottom strand as template 5’ GAUUACA 3’ 3’ CTAATGT 5’ 5’ GAUUACA 3’ Purves 4.9: proteins package DNA into chromosomes H. Sive MIT 2007 7 Purves 9.7: DNA is wrapped with proteins into chromatin which inhibits transcription The answer lies in The Promoter See Purves 14.12, 14.13 Question 3 How is chromatin unwrapped where transcription is needed? 8 9 The Promoter 5’Promoter non-template strand (“RNA-like”)3’ 5’ 3’ template strand Promoter = DNA sequence that indicates where transcription should begin and in what direction. gene 5’ 3’ transcription 3’ RNA 5’ start site H. Sive MIT 2007 Transcription initiation gene 5’ 3’ template strand 5’ Transcription factors Chromatin-associated recognize and bind promoter proteins removed/changed and DNA is “unwrapped” 3’ RNA 5’ 3’ start sitetemplate strand transcription Additional factors + RNA polymerase bind promoter (= initiation complex). DNA denatures locally and transcription begins H. Sive MIT 2007 10 11 Initiation complex: note relative sizes TATA binding protein (TBP) bound asymmetrically to DNA (TATAAA) 12 13 Transcription elongation and termination 3’ Parts of a gene… RNA transcription elongation 5’ Promoter 3’ termination 3’ transcript release Specific proteins bind and form a “Termination Complex” 5’ stop site Untranscribed (regulatory sequence) 3’ transcribed Untranscribed (regulatory sequence) 5’ H. Sive MIT 2007 H. Sive MIT 2007 14 Question 4 Not all genes are transcribed in every cell. Why? One answer: Some transcription factors are only found in certain cell types. Cell type Muscle Pancreas Factor present? MyoD Hnf4! yes no no yes 3. RNA processing Splicing Target gene transcribed? m-actin insulin yes no no yes 16 gene 15 See Purves 4.4, 14.10 Splicing transcription exon intron primary 5’ (“nascent”) RNA cytoplasm mature mRNA Prof. Phillip Sharp, MIT Nobel Prize 1993 “split genes” 3’ Splicing = introns removed “lariats” 5’ 3’ Gene includes exons and introns Introns = in primary RNA/ non-coding Exons = in mature mRNA/ protein coding H. Sive MIT 2007
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