('15nucbm'; Peter.Cook@path.ox.ac.uk; http://users.path.ox.ac.uk/~pcook)
The material covered in this lecture, coupled with the recommended reading, should enable you to:
• appreciate the size of nuclei and the different structures in it
• describe the different structures (types of chromatin, membrane, pores, nucleolus)
• understand how long DNA molecules are packed into nuclei
• describe the crucial nuclear functions (replication, transcription, repair, recombination)
Most conspicuous organelle in cell; diameter 2-10 micrometer (Fig); arguably most important (contains genome).
Function: contain/protect genome, carry out replication, transcription, repair, recombination, ribosome production.
Genes surrounded by envelope of 2 membranes.
Nuclear pores pierce envelope and allow communication between nucleus and cytoplasm.
Generally little internal structure visible in LM, apart from nucleoli (where rRNA synthesized, ribosomes assembled).
Most of rest filled with chromatin, recognized by reaction with basic stains (eg methylene blue, hematoxylin). Cellular components that bind basic and acidic dyes are termed basophilic and acidophilic.
Chromatin commonly divided into 2 types: euchromatin (dispersed, occupies most nuclear volume), heterochromatin (densely packed, structure more like that in mitotic chromosomes; Fig). Heterochromatin often condensed against envelope/nucleolus, can aggregate into densely-staining - often internal - chromocentres.
2 kinds of heterochromatin: constitutive (never expressed, often contains short DNA repeats), facultative (expressed in some cell lineages) -- example is X chromosome in female cells of mammals where one X (selected randomly early during development) is heterochromatic, (almost entirely) transcriptionally inactive, forms dense Barr body under nuclear membrane whilst the other (with essentially same DNA sequence) is euchromatic and inactive.
Size and structure
Typical human nucleus: ~10 µm diameter; occupies small fraction cell (6% vol in liver where 22% is mitochondrial).
Nuclei containing different amounts DNA may have roughly same size, conversely, those with same amount DNA can have very different sizes (eg nucleus in resting human lymphocyte is ~5 µm dia, in migratory neuroblast >20 µm).
DNA in a human chromosome is arguably the longest and most important biomolecule (Fig)
Widely assumed there are ~3,000,000,000 bp (or 3,000 Mbp) in a haploid human nucleus.
Typical human chromosome contains ~100 Mbp DNA; 2 nm wide, 3.4 cm long when stretched out; 3,000 Mbp DNA stretches 2 m. [From number of bp x distance between two in Watson-Crick structure (ie ~0.34 nm).]
Dimensions of molecules with such length-to-width ratios are far outside anything found in everyday world. Analogy: kite string (~2 mm dia) is a millionfold wider than DNA fibre - if had same length-to-width ratio as 100 Mbp of DNA, it would be 30 km long! String equivalent to 6000 Mbp in diploid nucleus stretches 2000 km (London-Rome).
Packing problem - reduce contour length ~10,000x. Easy to imagine packing controls access polymerases to DNA.
DNA strong acid - neutralized - histones H1, H2A/B, 3/4 (contain positively charged amino acids - lys, arg).
Histones highly conserved (2 differences in aa sequence of H4 from peas, cows).
Non-histone chromatin proteins (many bind to specific DNA sequences).
DNA coiled around nucleosome, into solenoid (?), loops, chromosome domains/territories (Fig).
Nuclear envelope - distinguishes eu- from pro-karyotes (Fig). Double membrane (inner, outer) separated by 20-40 nm (perinuclear space). In EM, outer membrane continuous with that of endoplasmic reticulum (ER); often outer surface studded with ribosomes (rough ER). Space between nuclear membranes continuous with lumen of ER.
A nuclear lamina - determines nuclear shape - underlies nucleoplasmic side of inner membrane. Fibrous mesh of lamin proteins, members of intermediate filament family (family contains keratins, vimentin).
Nuclear pores - each surrounded by a nuclear pore complex - pierce membrane (Fig, Fig); gates allowing in/out.
Each pore enormous (~125 x 106 D; ~100 different polypeptides). 8 large protein granules arranged in a circle around hole (internal/external dia ~80/~120 nm); 8 fibrils attached to cytoplasmic side of the ring, 'basket' on other side.
Central hole: aqueous ~9-nm channel allowing diffusion through membrane (proteins >9 nm cannot diffuse in/out).
Traffic density very high - in human cells ~50 histone molecules + ~100 ribosomal proteins enter every min through each pore, while ~2 ribosomal subunits (dia ~15 nm) exit!
Functions - gene transcription, replication, repair, recombination.
Label newly-made RNA and DNA with appropriate precursors (eg uridine and UTP/thymidine and TTP, tagged with 3H, 32P, Br, biotin) to label nascent RNA/DNA. Note phosphorylated precursors cannot cross cell membrane.
Active polymerases concentrated in discrete transcription/replication 'foci' or 'factories' (Fig).
28, 18, 5S rRNA made in nucleolus, a ribosome-producing factory (Fig). The nucleolus is the most prominent cytological feature of nucleus, with high concn RNA but little DNA. Its size reflects activity - small in dormant cells, can swell to 25% nuclear vol in cells actively making protein.
Reference:Ch 4,5 Alberts, B. et al. (2014). 'Molecular Biology of the Cell'. 6th Ed. Garland. [see also PubMed].
Pollard, T.D., Earnshaw, W.C., and Lippincott-Schwartz,J. (2007). 'Cell Biology'. 2nd Ed. WB Saunders/Elsevier.
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The Theme of Structure and Function in Cells
Up until now, we have spent some time (OK, maybe lots of time…stop looking at us like that) describing the junk, er, different components you might expect to find in different kinds of cells. We have also spent a lit—lots of time talking about what each of these unique components do for the cell. That is, we have talked about their functions. Hopefully, by now, you have begun to notice that, in almost every case, the structure of a given cellular component has a lot to do with its function. In fact, one mantra of biology encapsulates this idea perfectly: "Structure dictates function" (you should probably memorize this phrase now). The name for these relationships are, uh, structure-function relationships. To really appreciate how true this idea is, let’s look at a few examples in detail.
Let's zip back to mitochondria and chloroplasts. These organelles are really nothing more than membranes within membranes, with a little space between said membranes. The main function of mitochondria is to convert the energy in glucose to ATP, a usable form of energy for the cell, through the process of cellular respiration. This exceedingly important function is only possible because of the unique structure of the mitochondrial membranes, which allow for an intermembrane space to form where protons can accumulate, and for a matrix to which the protons can flow.
Without the inner mitochondrial membrane, or IMM, there would be no "Hoover Dam" to hold back protons and force them to flow through the ATP synthase rotor. Moreover, the IMM is folded into structures called cristae, which pave the way for millions of ATP synthase complexes to jam into a single mitochondrion. Sounds a little crowded. Without the unique folded structure of cristae, cells would need millions of mitochondria in order to produce the same amount of energy produced by just a few with cristae. Structure dictates function.
As for chloroplasts, without the thylakoid membranes separating the stroma from the lumen, there would be no space for protons to accumulate and flow back into. Without the products produced by the thylakoid membrane proteins, including ATP (we know; he's everywhere), and without a space for glucose to be made, or the stroma, photosynthesis would not occur, and life on Earth as we know it would cease to exist. Are you ready to acknowledge the vital relationship between structure and function yet, or what? Do you want the world to end? DO you?!
In the end, only the structures of the mitochondria and chloroplasts allow the processes of cellular respiration and photosynthesis to take place. In both cases, the presence of a membrane allows for compartments to form. Those compartments can have different concentrations of hydrogen ions, and it is those differences in concentration that drive formation of important substances.
Ribosomes provide another good example of structure determining function. These small cellular components are made of protein and ribosomal RNA (rRNA). Their main function is to translate messenger RNA, or mRNA, into strings of amino acids called proteins.
Ribosomes are composed of two main parts:
- A large subunit
- A small subunit.
Let's go back to our picture of a complete ribosome:
The small subunit has a special groove that allows for mRNA to bind to it. Once the mRNA is bound, the large subunit attaches on top, and a complete ribosome is formed. mRNA is pulled through the space between the two subunits as another molecule, transfer RNA (tRNA), binds to a second groove in the ribosome and to the mRNA, leaving behind an amino acid in yet a third groove.
For every three base pairs of mRNA, tRNA leaves behind one specific amino acid. When the end of the mRNA strand is reached, the ribosome subunits detach and let both the mRNA and the newly formed string of amino acids, aka the protein, run free into the big wide world. The grooves of the ribosome allow for mRNA to be held in place while tRNA reads the "code" that determines which amino acid is next in the sequence. It is the very structure of ribosomes that completes the Central Dogma of Biology, or DNA to RNA to Protein.
Without proteins, a big, fat nothing would get done in the cell. N.O.T.H.I.N.G.