Lecture 5
Lecture 5 September-22-12 Review:
Reassociation is inversely proportional to the genome (DNA) size(Refer to above photo) Mouse satellite - repetitive genes in the mouse genome - The DNA sources are: a synthetic DNA duplex of poly A and poly U polynucleotide chains; mouse satellite DNA, a fraction of mouse DNAin which the same sequence is repeated many thousands of times; - MS2 DNA (bacteriophage) - t4 DNA(more complex bacteriophage then MS-2) - E.coli DNA; calf DNA-nonrepetitive fraction (highly repetitive DNA fraction was removed) Cot analysis recently revived: aids genome sequencing - Unique, protein coding sequences could be 'purified' from the mixture - Faster then shotgun sequencing (repetitive sequences are not sequenced) - Cheaper then shotgun sequencing - Unique sequence is easier to sequence
Determining Genome Size by Cot Analysis
Is Complexity of genome in correlation with the biological complexity of the organism? N=haploid chromosome number
NOTES FROM LECTURES Page 1
N=haploid chromosome number C= DNA content/haploid cell C varies greatly among phyla General increase from prokaryotes to eukaryotes Large differences within eukaryotes - C-paradox; no correlation between amount of DNA in genome and apparent complexity ○ No correlation between the amount of DNA [size of genome] and the apparent complexity of organisms - More DNA present than we can account for… - Can you say...Increase in genome=increase in complexity
- Prokaryotic = nonrepetitive DNA - Proportions of different sequence types vary among eukaryotic genomes ○ Graph seperates unique from repetition Analyze trends in the red bars; proportions don’t stay the same → Can you see trends with nonrepetitive? → It seems to increase then coming to a plateu...absolute content of non-repetitive DNA increases with genome size; plateaus at 2x10^9bp
(Lewin, 2006. Fig. 4.9)
Evolution of biological complexity/genome increase as a clock for the origin and evolution of life - New definition of biological complexity = size of functional and non-repetitive section of a genome ○ Duplicated, inserted etc. sequences are not included in 'complexity calculations' - this is based on the idea that the unique portion is "running the show" - Hypothesis: increase in size of the unique part of a genome is due to the positive feedback mechanisms during evolution: 1. Already present genes [including genes which improve proof-reading during replication] help establishment [survival] of new genes [bigger genomes grow faster] 2. Big genomes provide more options for recombinations and duplications, which leads to creation of new genes [bigger genomes grow faster] eg. Plants and protists have so much DNA because there environment changes all the type so they need to be able to adapt correctly 3. Complex metabolic pathways and complex body structure [found in higher organisms] require more protein coding genes - question of efficiency [bigger genomes grow faster] Circular DNA - Prokaryotic genomic DNA and viral DNAs are circular - Circular genome is composed of two strands of DNA that form a closed structure without free ends (double circle) - Chloroplast and mitochondria also have a circular genome ○ Endosymbiotic theory; originally a bacteria that supported itself ○ However, now they are unable to live on there own and cannot support themselves
NOTES FROM LECTURES Page 2
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Denaturation of Circular DNA - Circular DNA can also be denatured like linear DNA - However, two strands cannot unwind and separate like linear DNA when the bases come apart ○ In vivo, nicking occurs naturally during DNA replication; one strand only is nicked and the other stays circular
○ Can be induced experimentally by using an enzyme Both Circular and Linear DNA: - Primary structure of DNA: sugar-phosphate "chain" with purine and pyrimidine bases as side chain(s) - Secondary Structure of DNA double helical structure [hydrogen bonding between A-T and G-C; stacking interactions; phosphate backbone 'outside'] - Tertiary or higher order structure double stranded DNA [both circular AND linear] makes complexes with proteins possible for it to form a supercoil - Supercoiling = coiling of a coil Supercoils - Important for packing of DNA - DNA helix becomes topographically linearized [local uncoiled] during replication and transcription - Topological isomers - DNA differing only in their states of supercoiling; different forms that only differ in there conformation **critical for understanding how DNA can be packed efficiently and still be accessible; must have bases accessible for transcription and translation
Conformations of RNA Different types/functions of RNA: - mRNA: messenger RNA, specifies order of amino acids during protein synthesis - tRNA: transfer RNA, during translation mRNA information is interpreted by tRNA ○ Nucleic acid to amino acid 'translater' - rRNA: ribosomal RNA, combined with proteins aids tRNA in translation
NOTES FROM LECTURES Page 3
- rRNA: ribosomal RNA, combined with proteins aids tRNA in translation ○ Acts as an enzyme in cell; critical for translation - RNAs with enzymatic functions; ribozymes ○ Involved in splicing and peptide bond formation during protein synthesis - Small RNAs - different functions Note: the conformation is important for function
Main RNA; code for something *all stages are susceptible to changes from other RNA - RNAs have to be produced correctly therefore multiple regulations
Central Dogma meets more RNA - The original one was done by Francis Crick - Revised central dogma
Conformations of RNA - Primary structure of RNA similar to DNA ○ 2' OH group prevents formation of B-helix: A-helix is formed ○ RNA can be, single or double; linear or circular ○ Unlike DNA, RNA can exhibit different conformations ○ Different conformations (2prime and 3prime structure formation) permit the RNAs to carry out specific functions in the cell ; more functions then that of DNA
Secondary Structure in RNA:
NOTES FROM LECTURES Page 4
Secondary Structure in RNA: - RNA molecule frequently fold back on themselves to form base-paired segments between short stretches of complementary sequences - G:U = additional, non-Watson&Crick base pairing possible in RNA --> enhances potential for self-complementary in RNA ○ Can also have heavily modified bases - Secondary structures: areas of regular helices and discontinuous helices with stem-loops [>50 nucleotides] or hairpins[4-10 nucleotides] ○ Short segments base pair and create these weird shapes - Has major and minor grooves present but they are not dominant
Tertiary Structure in RNA: - Formed through interactions of secondary structure: ○ Lack of constraint by long-range regular helices means RNA has a high degree of rotational freedom in backbone of its non-base-paired regions Capable of folding into complex tertiary structures The ss has high ability for mobility - Below; picture of a tRNA which is 'Lshape'
NOTES FROM LECTURES Page 5
- Formation of unconventional triple base pairing is possible ○ A little unusual because they done follow the rules Ability of hydrogens to take part Donor and acceptor [solide line=donating and dashed=accepting]
- Pseudoknots can form due to base-pairing sequences that are not adjacent ○ Multiple areas in ss that take part in weird base pairings
-
Note: Intron don’t get coded for can be enzymes - Cells are super efficient...not junk! - Example of a giant tertiary RNA...
NOTES FROM LECTURES Page 6
Example of a giant tertiary RNA...
Tetrahymena Group I intron (ribozyme) secondary and tertiary structures; from: http://academic.brooklyn.cuny.edu/chem/zhuang/QD/toppage1.htm
NOTES FROM LECTURES Page 7
Reassociation is inversely proportional to the genome (DNA) size(Refer to above photo) Mouse satellite - repetitive genes in the mouse genome - The DNA sources are: a synthetic DNA duplex of poly A and poly U polynucleotide chains; mouse satellite DNA, a fraction of mouse DNAin which the same sequence is repeated many thousands of times; - MS2 DNA (bacteriophage) - t4 DNA(more complex bacteriophage then MS-2) - E.coli DNA; calf DNA-nonrepetitive fraction (highly repetitive DNA fraction was removed) Cot analysis recently revived: aids genome sequencing - Unique, protein coding sequences could be 'purified' from the mixture - Faster then shotgun sequencing (repetitive sequences are not sequenced) - Cheaper then shotgun sequencing - Unique sequence is easier to sequence
Determining Genome Size by Cot Analysis
Is Complexity of genome in correlation with the biological complexity of the organism? N=haploid chromosome number
NOTES FROM LECTURES Page 1
N=haploid chromosome number C= DNA content/haploid cell C varies greatly among phyla General increase from prokaryotes to eukaryotes Large differences within eukaryotes - C-paradox; no correlation between amount of DNA in genome and apparent complexity ○ No correlation between the amount of DNA [size of genome] and the apparent complexity of organisms - More DNA present than we can account for… - Can you say...Increase in genome=increase in complexity
- Prokaryotic = nonrepetitive DNA - Proportions of different sequence types vary among eukaryotic genomes ○ Graph seperates unique from repetition Analyze trends in the red bars; proportions don’t stay the same → Can you see trends with nonrepetitive? → It seems to increase then coming to a plateu...absolute content of non-repetitive DNA increases with genome size; plateaus at 2x10^9bp
(Lewin, 2006. Fig. 4.9)
Evolution of biological complexity/genome increase as a clock for the origin and evolution of life - New definition of biological complexity = size of functional and non-repetitive section of a genome ○ Duplicated, inserted etc. sequences are not included in 'complexity calculations' - this is based on the idea that the unique portion is "running the show" - Hypothesis: increase in size of the unique part of a genome is due to the positive feedback mechanisms during evolution: 1. Already present genes [including genes which improve proof-reading during replication] help establishment [survival] of new genes [bigger genomes grow faster] 2. Big genomes provide more options for recombinations and duplications, which leads to creation of new genes [bigger genomes grow faster] eg. Plants and protists have so much DNA because there environment changes all the type so they need to be able to adapt correctly 3. Complex metabolic pathways and complex body structure [found in higher organisms] require more protein coding genes - question of efficiency [bigger genomes grow faster] Circular DNA - Prokaryotic genomic DNA and viral DNAs are circular - Circular genome is composed of two strands of DNA that form a closed structure without free ends (double circle) - Chloroplast and mitochondria also have a circular genome ○ Endosymbiotic theory; originally a bacteria that supported itself ○ However, now they are unable to live on there own and cannot support themselves
NOTES FROM LECTURES Page 2
○
Denaturation of Circular DNA - Circular DNA can also be denatured like linear DNA - However, two strands cannot unwind and separate like linear DNA when the bases come apart ○ In vivo, nicking occurs naturally during DNA replication; one strand only is nicked and the other stays circular
○ Can be induced experimentally by using an enzyme Both Circular and Linear DNA: - Primary structure of DNA: sugar-phosphate "chain" with purine and pyrimidine bases as side chain(s) - Secondary Structure of DNA double helical structure [hydrogen bonding between A-T and G-C; stacking interactions; phosphate backbone 'outside'] - Tertiary or higher order structure double stranded DNA [both circular AND linear] makes complexes with proteins possible for it to form a supercoil - Supercoiling = coiling of a coil Supercoils - Important for packing of DNA - DNA helix becomes topographically linearized [local uncoiled] during replication and transcription - Topological isomers - DNA differing only in their states of supercoiling; different forms that only differ in there conformation **critical for understanding how DNA can be packed efficiently and still be accessible; must have bases accessible for transcription and translation
Conformations of RNA Different types/functions of RNA: - mRNA: messenger RNA, specifies order of amino acids during protein synthesis - tRNA: transfer RNA, during translation mRNA information is interpreted by tRNA ○ Nucleic acid to amino acid 'translater' - rRNA: ribosomal RNA, combined with proteins aids tRNA in translation
NOTES FROM LECTURES Page 3
- rRNA: ribosomal RNA, combined with proteins aids tRNA in translation ○ Acts as an enzyme in cell; critical for translation - RNAs with enzymatic functions; ribozymes ○ Involved in splicing and peptide bond formation during protein synthesis - Small RNAs - different functions Note: the conformation is important for function
Main RNA; code for something *all stages are susceptible to changes from other RNA - RNAs have to be produced correctly therefore multiple regulations
Central Dogma meets more RNA - The original one was done by Francis Crick - Revised central dogma
Conformations of RNA - Primary structure of RNA similar to DNA ○ 2' OH group prevents formation of B-helix: A-helix is formed ○ RNA can be, single or double; linear or circular ○ Unlike DNA, RNA can exhibit different conformations ○ Different conformations (2prime and 3prime structure formation) permit the RNAs to carry out specific functions in the cell ; more functions then that of DNA
Secondary Structure in RNA:
NOTES FROM LECTURES Page 4
Secondary Structure in RNA: - RNA molecule frequently fold back on themselves to form base-paired segments between short stretches of complementary sequences - G:U = additional, non-Watson&Crick base pairing possible in RNA --> enhances potential for self-complementary in RNA ○ Can also have heavily modified bases - Secondary structures: areas of regular helices and discontinuous helices with stem-loops [>50 nucleotides] or hairpins[4-10 nucleotides] ○ Short segments base pair and create these weird shapes - Has major and minor grooves present but they are not dominant
Tertiary Structure in RNA: - Formed through interactions of secondary structure: ○ Lack of constraint by long-range regular helices means RNA has a high degree of rotational freedom in backbone of its non-base-paired regions Capable of folding into complex tertiary structures The ss has high ability for mobility - Below; picture of a tRNA which is 'Lshape'
NOTES FROM LECTURES Page 5
- Formation of unconventional triple base pairing is possible ○ A little unusual because they done follow the rules Ability of hydrogens to take part Donor and acceptor [solide line=donating and dashed=accepting]
- Pseudoknots can form due to base-pairing sequences that are not adjacent ○ Multiple areas in ss that take part in weird base pairings
-
Note: Intron don’t get coded for can be enzymes - Cells are super efficient...not junk! - Example of a giant tertiary RNA...
NOTES FROM LECTURES Page 6
Example of a giant tertiary RNA...
Tetrahymena Group I intron (ribozyme) secondary and tertiary structures; from: http://academic.brooklyn.cuny.edu/chem/zhuang/QD/toppage1.htm
NOTES FROM LECTURES Page 7
