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Advanced Cell Biology. Lecture 12
Advanced Cell Biology. Lecture 12
Alexey Shipunov
Minot State University
February 10, 2012
Advanced Cell Biology. Lecture 12
Outline
Questions and answers
ProteinsHow proteins are studied
DNADNA replication
Advanced Cell Biology. Lecture 12
Outline
Questions and answers
ProteinsHow proteins are studied
DNADNA replication
Advanced Cell Biology. Lecture 12
Outline
Questions and answers
ProteinsHow proteins are studied
DNADNA replication
Advanced Cell Biology. Lecture 12
Questions and answers
Previous final question: the answer
Which way of sedimentation employs differences betweenmolecular sizes?
I Velocity sedimentation: larger proteins sediment faster
Advanced Cell Biology. Lecture 12
Questions and answers
Previous final question: the answer
Which way of sedimentation employs differences betweenmolecular sizes?
I Velocity sedimentation: larger proteins sediment faster
Advanced Cell Biology. Lecture 12
Questions and answers
Teams
I Studies on the chemical nature of the substance inducingtransformation of Pneumococcal types. — Avery, O.T.,MacLeod, C.M. & McCarty, M.J. — Exp. Med. 79, 137-159(1944)
I Molecular Structure of Deoxypentose Nucleic Acids —Wilkins M.H.F., A.R. Stokes A.R. & Wilson, H.R. —Nature171, 738-740 (1953)
I (1) A Structure for Deoxyribose Nucleic Acid — WatsonJ.D. and Crick F.H.C. — Nature 171, 737-738 (1953) AND(2) Genetical Implications of the structure ofDeoxyribonucleic Acid — Watson J.D. and Crick F.H.C. —Nature 171, 964-967 (1953)
Advanced Cell Biology. Lecture 12
Proteins
How proteins are studied
ProteinsHow proteins are studied
Advanced Cell Biology. Lecture 12
Proteins
How proteins are studied
X-rays crystallography
I Based on the ability of proteins to form crystalsI These crystals will scatter X-rays and we will see difraction
pattern, different for different proteinsI Useful for prediction of protein 3D structure
Advanced Cell Biology. Lecture 12
Proteins
How proteins are studied
X-rays crystallography
Advanced Cell Biology. Lecture 12
Proteins
How proteins are studied
Nuclear magnetic resonance (NMR)
I Unfortunately, not all proteins form crystals and it is notalways possible to obtain enough protein for crystallization
I Method is based on the response of nuclei to radio wavesI Again, it is used mostly for understanding conformation of
protein
Advanced Cell Biology. Lecture 12
Proteins
How proteins are studied
Nuclear magnetic resonance
Advanced Cell Biology. Lecture 12
Proteins
How proteins are studied
Protein databases
I UniProtI NCBI proteinI RCSB
Advanced Cell Biology. Lecture 12
Proteins
How proteins are studied
NCBI Protein database
Advanced Cell Biology. Lecture 12
Proteins
How proteins are studied
RCSB database
Advanced Cell Biology. Lecture 12
Proteins
How proteins are studied
UniProt database
Advanced Cell Biology. Lecture 12
DNA
DNA replication
DNADNA replication
Advanced Cell Biology. Lecture 12
DNA
DNA replication
Template
I Both DNA strands may act as a template for the synthesisof other strand
I Template hypothesis was first expressed by Nikolaj Koltsovin 1927
I Replication is semiconservative
Advanced Cell Biology. Lecture 12
DNA
DNA replication
Advanced Cell Biology. Lecture 12
DNA
DNA replication
Advanced Cell Biology. Lecture 12
DNA
DNA replication
Advanced Cell Biology. Lecture 12
DNA
DNA replication
Meselson-Stahl experiment (1958)
I Theoretically, three variants of replications were possible:semiconservative, dispersive and conservative
I Experiment was based on two bacterial cultures whichgrew on different media: with normal nitrogen 14N, andwith heavy nitrogen, 15N
I After growing for 20’ on heavy medium, bacteria produceDNA molecules with intermediate weight only
I That ruled out conservative hypothesis.
How to rule outdispersive hypothesis?—Explanation has been given onlecture.
Advanced Cell Biology. Lecture 12
DNA
DNA replication
Meselson-Stahl experiment (1958)
I Theoretically, three variants of replications were possible:semiconservative, dispersive and conservative
I Experiment was based on two bacterial cultures whichgrew on different media: with normal nitrogen 14N, andwith heavy nitrogen, 15N
I After growing for 20’ on heavy medium, bacteria produceDNA molecules with intermediate weight only
I That ruled out conservative hypothesis. How to rule outdispersive hypothesis?—Explanation has been given onlecture.
Advanced Cell Biology. Lecture 12
DNA
DNA replication
Advanced Cell Biology. Lecture 12
DNA
DNA replication
Replication origin and forks
I DNA double helix should open for replication: this is areplication origin place
I When replication starts, these openings will grow and formreplication forks which are visible under microscope
Advanced Cell Biology. Lecture 12
DNA
DNA replication
Advanced Cell Biology. Lecture 12
DNA
DNA replication
Advanced Cell Biology. Lecture 12
DNA
DNA replication
Lagging and leading strands
I DNA is synthesized only in 5’-to-3’ direction. Structureof DNA and complexity of replication do not allow the otherdirection.
I Therefore, fork is asymmetrical: one strand is replicatingsmoothly whereas other strand (lagging) replicated by“leaps”, still 5’-to-3’
I Every “leap” produce one Okazaki fragment which are laterjoined
Advanced Cell Biology. Lecture 12
DNA
DNA replication
Advanced Cell Biology. Lecture 12
DNA
DNA replication
DNA proofreading
I Every time DNA polymerase adds new nucleotide, itchecks if previous was correctly placed (if not, it removesthe wrong nucleotide)
I Two different binding sites in DNA polymerase work forsynthesis and proofreading
I Proofreading goes in opposite, 3’-to-5’ directionI As a result, DNA polymerase enzyme error rate became
less than ≈0.00000001
Advanced Cell Biology. Lecture 12
DNA
DNA replication
Advanced Cell Biology. Lecture 12
DNA
DNA replication
RNA primers
I DNA polymerase cannot start nucleotide chain itselfI Instead, primase enzyme synthesize small RNA primer
(≈10 nucleotides) which used as a starting pointI Primase error rate is ≈0.0001 (!)I In lagging strand, RNA sites are interleaving with DNA
fragmentsI Then RNA sites are erasing, and DNA ligase joins
fragments together
I Why primer is RNA?
Advanced Cell Biology. Lecture 12
DNA
DNA replication
RNA primers
I DNA polymerase cannot start nucleotide chain itselfI Instead, primase enzyme synthesize small RNA primer
(≈10 nucleotides) which used as a starting pointI Primase error rate is ≈0.0001 (!)I In lagging strand, RNA sites are interleaving with DNA
fragmentsI Then RNA sites are erasing, and DNA ligase joins
fragments togetherI Why primer is RNA?
Advanced Cell Biology. Lecture 12
DNA
DNA replication
Advanced Cell Biology. Lecture 12
DNA
DNA replication
Final question (3 points)
Why cells use RNA as DNA replication primers?
Advanced Cell Biology. Lecture 12
DNA
DNA replication
Final question (3 points)
Why cells use RNA as DNA replication primers?
Advanced Cell Biology. Lecture 12
DNA
DNA replication
Summary
I DNA replication is a semiconservative processI DNA replication could go only in one directionI Proofreading and RNA priming are helping in replication
Advanced Cell Biology. Lecture 12
DNA
DNA replication
For Further Reading
A. Shipunov.Advanced Cell Biology [Electronic resource].2011—onwards.Mode of access: http://ashipunov.info/shipunov/school/biol_250
B. Alberts et al.Essential Cell Biology. 3rd edition.Garland Science, 2009.Chapter 6, pages 198–208.