EFB325 Cell Physiology

The Central Dogma

The third section of the course will describe the flow of information in cells. This includes the information and mechanism needed to determine the sequences of proteins, which proteins will accumulate in particular cells, how the levels of individual proteins can change in response to stimuli or during development, and also, how this information is copied and passed on to daughter cells.

The central dogma

We now know the "path" of information in cells, including the inherited genetic material and the mechanism of converting that information into functional proteins.

DNA=stores information;

=>is copied (accomplished by DNA polymerase) and transmitted during cell division and reproduction

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    | =transcription (synthesis of RNA using a DNA template accomplished by RNA polymerase)
   V

RNA=intermediate in the conversion of information in DNA to functional protein; conversion process can be regulated by varying the rate of RNA synthesis

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    | =translation (synthesis of protein accomplished by ribosomes)
   V

protein=functional components of cell metabolism; contain information to direct them to proper cellular location; protein-protein interactions can regulate metabolism

How do we know that DNA is the genetic material?

Oswald Avery and colleagues (early '40's) determined that DNA had the ability to transform bacteria

• used two strains of Pneumococcus bacteria; smooth=killed mice, rough=mice lived
• heat kill smooth strain=mice lived
• living rough + heat-killed smooth=mice killed
• compounds from heat-killed smooth bacteria were fractionated and added to rough cells
• DNA was found to be the active component able to transform the rough strain cells

Alfred Hershey and Martha Chase (early '50's) determined that bacterial viruses (bacteriophage) injected DNA into their host cells

• used radiolabeled virus particles= ³²P used to label DNA, ³⁵S used to label protein coat
• upon infection of bacteria, the ³²P (as a component of DNA) was mostly inside the cell (which became infected with the virus); the ³⁵S (in the protein) always stayed in the liquid

DNA-structure and function

How is DNA organized in the cell?

In prokaryotes, mitochondria, and chloroplasts: there is one circular chromosome (but there may be multiple copies of that chromosome per cell or per organelle)

In eukaryotes: chromosomes are very long linear molecules

The amount of DNA in an organism's genome shows general trends, but can be extremely variable between closely related organisms

• viruses have the least DNA
• bacteria have more
• fungi still more
• all other eukaryotes have more DNA than any bacterium
• genomes size is quite variable within taxa

Chromosomes

The DNA of a single human cell is about 1 m in length. The DNA of E. coli is about 1.6 mm, and the DNA of a single Trillium cell is about 34 m in length.

So how does such a long molecule fit into a nucleus that may be only 8-10 micrometers in diameter?

Packaging of chromosomes

In bacteria:

• the circular chromosome is supercoiled (like a twisting a coiled phone cord), then folded into loops

In eukaryotes:

• there are multiple levels of organization
• packing is coordinated by proteins that associate with DNA

Levels of packing in a eukaryote

1) DNA strand wraps around a core of histone proteins to form a nucleosome

• histones are high in the amino acids lysine and arginine, which have a positive charge that interacts with the negative charge on DNA
• approx. 150 bp of DNA per nucleosome
• there is an unwound region of spacer DNA between the nucleosomes (~50 bp)

2) Nucleosomes pack/wrap together to form the 30 nm chromatin fiber

• chromatin describes the combination of DNA and proteins, which together form the chromosomes

3) The 30 nm chromatin fibers form large loops=looped domains

• looped domains are the sites of active gene expression (transcription)
• each loop contains ~50,000 - 100,000 bp of DNA

4) densely packed looped domains=heterochromatin

• represents regions of low transcriptional activity
• chromatin packs during mitosis
• represents 15,000-20,000-fold packing

The nucleus has structural features related to its function

nucleolus=site where long stretches of chromosome that encode the rRNA (ribosomal RNA) are actively transcribed; ribosomal proteins enter the nucleus and are combined with the rRNA to make new ribosome subunits

nuclear envelope=nucleus is surrounded by two membranes

• the outer membrane is continuous with the ER membrane (space between the membranes is continuous with the ER lumen)
• the inner membrane is associated with a layer of structural proteins on the inside that keep the shape of the nucleus=lamina

nuclear pores=large complex of proteins that allow/regulate transport in and out of the nucleus

• proteins enter from the cytoplasm (histones, ribosomal proteins, enzymes for DNA replication & transcription, lamina proteins, etc.)
• RNA crosses out to the cytoplasm=ribosomal RNA (rRNA), transfer RNA (tRNA), and messenger RNA (mRNA)

What is the structure of DNA and how is that related to function?

Erwin Chargaff (early '50's) discovered that an organism's DNA always contains equal amounts of pyrimidines and purines (A+G=C+T)=Chargaff's rules

• used chemistry to determine the % of A, C, G, or T in an organism's DNA
• among individuals of the same species, the % were always the same
• between different species, the % were variable, but always %A=%T, %G=%C

James Watson and Francis Crick (1953) resolved the structure of DNA (using data obtained by Rosalind Franklin) as a double helix

• Franklin had used x-ray diffraction to determine that there were two strands
• Watson and Crick determined that pyrimidines were pairing with purines between two anti-parallel strands
• minor groove=space between base-paired strands; major groove=space due to helix

The discovery that DNA exists in two complementary strands provided a basis to explain the mechanism of storage and transmission of genetic information to daughter cells by DNA replication.

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