229.DNANucleas103116 (1) - Structural basis of Cellular...

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Unformatted text preview: Structural basis of Cellular Informa3on: DNA, chromosomes and the nucleas 18 10/31/16 Storage, transmission and decoding of informa3on Transforming principle: heat stable carrier of gene3c informa3on. •  •  •  •  Live S but not R bacteria can cause pneumonia Live R bacteria mixed with heat killed S bacteria can also pneumonia. Bacteria recovered from the second experiment were – what? What could be concluded from this experiment ? DNA: Carrier of gene3c informa3on. Chargaffs rules and the chemical composi3on of DNA. •  DNA is composed of a sugar (deoxy ribose) phosphate backbone and nitrogenous bases (purines and pyramidines). •  In any organism the A,T, G and C composiQon is invariant between cells but different between organisms. •  Total number of purines is equal to the number of pyrimidines. •  The raQo of A to T and G to C is always 1, but not other raQos. The double helix: the physical structure of DNA. •  X-­‐ray diffrac3on studies revealed key parameters of the structure. •  DNA is a narrow helix 2 nm in width. •  One structurally repeated unit of 3.4 nm corresponds to one turn of DNA. The double helix: the physical structure of DNA. •  The deoxy ribose-­‐ phosphate chain forms the backbone. The bases project inward to form a twisted ladder-­‐like structure. •  The double helix is held together by H bonds. 2 between A and T and three between G and C. •  The helix is right handed with major and minor grooves. The bases in the major groove are more “exposed”. The double helix-­‐ contd . •  •  •  The 2 strands are anQ-­‐parallel The 5’ end of one strand pairs with the 3’ end of the other strand. DNA exists mainly as a B helix (right-­‐handed) but someQmes as a le] handed Z helix. Mel3ng or separa3on of strands and annealing. Genome size and complexity. •  •  •  Genome size refers to the length in base pairs (bps) of the whole haploid genome Complexity refers to the extent of unique sequences in the genome. The majority of eukaryoQc DNA is comprised of repeQQve sequences. Annealing proper3es of DNA reveal the presence of repe33ve sequences. •  In general shorter genomes anneal faster than longer genomes at similar concentraQons. Why ? •  RepeQQve DNA sequences deviate from this behavior. •  Part of calf DNA anneals faster than bacterial DNA and part much slower. Discrepancies between genome size and number of genes. •  Genome analysis of eukaryotes vs bacteria show that size does not correlate with gene number (103 vs. ~10 fold). •  Only <2% of the human genome encodes for proteins. •  What does the remaining 98% encode? The majority of the genome is non-­‐coding and repe33ve. •  A small part of the non-­‐coding region region performs regulatory funcQons. •  Majority of it is made up of short (1 bp) or long repeat (8000 bp) elements . The majority of the genome is non-­‐coding and repe33ve. •  These elements are present as tandem or interspersed elements. •  These sequences may impart special physical properQes to certain regions of chromosomes (tandem repeats). •  Interspersed elements may facilitate evoluQonary adaptability. Packaging of DNA. •  Genomic DNA of most cells is 500-­‐15,000 Qmes longer than the cell or nucleas. •  It has to be packaged to be both compact and accessible. •  Bacterial DNA is not associated with many proteins Bacterial DNA is looped and segregated into a nucleoid structure. Packaging of DNA. •  Genomic DNA of most cells is 500-­‐15,000 Qmes longer than the cell or nucleas. •  EukaryoQc DNA exists as “chromaQn” which consists of equal parts of DNA and protein. Packaging and organiza3on of eukaryo3c DNA. •  Genomic DNA is packaged into 10 nm fibers resembling “beads on a string” •  By wrapping around protein parQcles composed of histone proteins. •  Limited nuclease digesQon reveals a packaging length of 200bp. Nucleosome: primary units of chroma3n. •  A nucleosome consists of 146 bp wrapped around a histone octamer (composed of 2 molecules of histones H2A, H2B, H3 and H4). •  Adjacent nucleosomes are separated by a ~50 bp linker and connected by the linker histone H1 . •  This produces a 10 nm diameter fiber and consQtutes the first order of chromaQn. Higher orders of chroma3n compac3on. Chroma3n compac3on and DNA ac3vity. •  The extent of chromaQn compacQon is inversely related to the acQvity of the associated DNA . •  TranscripQonally acQve regions of DNA are loosely packaged into euchroma3n composed of 30 nm fibers and DNA loops. •  In interphase cells, regions of DNA Qghtly packaged into heterochroma3n are transcripQonally inacQve and electron dense. •  HeterochromaQn is mostly comprised of repeQQve DNA sequences. Nuclear compartmentaliza3on of the genome. Nuclear pores control nuclear cytoplasmic traffic. •  The nucleas is segregated from the cytoplasm by a double layered nuclear envelope which is conQguous with the ER. •  Molecules <10 nm in size can diffuse between the nucleas and cytoplasm. •  The movement of larger molecules is regulated by the nuclear pore complex . Export and import from and to the nucleas. Mechanism of nuclear protein import. •  Proteins imported into the nucleas have a nuclear localiza3on signal. •  The NLS is recognized and bound by impor3n which brings it through the NPC. •  Nuclear Ran(GTP) associates with imporQn and releases the cargo protein. •  The imporQn-­‐Ran(GTP) complex translocates to the nucleas where GTP is hydrolyzed. Mechanism of nuclear protein export. •  The nucleas exports RNA, proteins, and ribonucleoprotein complexes. •  RNA is bound by RNA binding proteins bearing nuclear export signals. •  NES is recognized by exporQn and Ran(GTP) and the complex transported across the NPC. Mechanism of nuclear protein export -­‐ 2. •  The cargo RNA and protein are released in the cytoplasm. •  ExporQn shuiles back to the nucleas. •  Following GTP hydrolysis Ran(GDP) is translocated back to the nucleas by NTF2. Structure and support of the nucleas. •  Structural integrity and support of the nucleas is provided by a scaffold made of lamin fibers. •  The nuclear lamina also provides aiachment for specialized regions of the genome i.e. heterochromaQn. Nucleolus: ribosome factories. •  Nucleoli are darkly staining regions of the nucleas where ribosomes are synthesized and assembled. ...
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