(Please visit the following web site to complement this lecture: http://cellbio.utmb.edu/cellbio/nucleus.htm)

1. Describe the structure of a eucaryotic nucleus, Alberts et al, Fig 8-1.
2. Why a nucleus?

• To protect the DNA molecules from the shearing forces generated by the cytoskeleton
• To segregate transcription from translation

3. Chromosomal DNA an its packaging

• In yeast, three types of DNA sequences, less than 1000 bp each, are required to ensure the propagation of chromosomes from generation to generation (Fig 8-4):

1. Centromere: 1/chromosome, holds together two copies of duplicated chromosomes, and attach them to mitotic spindle so that one copy goes to each daughter cell during mitosis.
2. Telomere: 2/chromosome, repeated sequence needed at the two ends of linear DNA to solve the "end-replication problem". Well characterized in humans too.
3. Replication origins, multiple sites along DNA where DNA replication starts.

• No relationship between amount of DNA in genome and organism complexity (Fig 8-6).
• A protein of average size has ~400 aa, i.e. encoded by a 1200 bp mRNA. About 10⁵ genes are thought to be encoded by the 3 x 10⁹ bp-human genome. Thus, only ~1.2 x 10⁸ bp, or less than 10% of the genome,codes for protein, hence, eucaryotic genomes carry much more DNA than what is needed to code for their estimated number of proteins. Thus, coding regions are named exons and non-coding regions are named introns. Genes are regions of the DNA helix that produce a functional RNA molecule (mRNA, rRNA or tRNA). Genes are made up of regulatory sequences (5' and 3') and exons and introns. The initial RNA transcribed from a gene is called a primary transcript. It is processed in various ways in the nucleus before being exported to the cytoplasm for translation (Fig 8-7). DNA sequence conservation between species allows identification of coding and non-coding regions between genomes.
• Chromatin = DNA + DNA-binding proteins. Two classes of DNA-binding proteins: nonhistone proteins and histones. Histones are highly-conserved, K-,R-rich small proteins. There are 5 types: two copies each of H2a, H2b, H3 and H4, make up the nucleosome's histone octamer, around which 146bp of DNA sequence is wrapped around (2 turns)à "beads-on-a-string" EM form (Figs 8-9, 8-10). Histones H1 (6 closely related subtypes in mammalian cells) are thought to be responsible for pulling nucleosomes together to form the 30-nm fiber (Figs 8-13 and 8-15). The linker DNA region between nucleosomes varies in length since nucleosome position is determined by the local flexibility of the DNA and by the distribution of other proteins bound to specific DNA sequences. Linker regions, covered with less complex proteins are revealed by treating cell nuclei with limiting amounts of DNAse I., which are readily digested by the enzyme. Such nuclease-hypersensitive sites often lie in the regulatory regions of genes.

4. The Global Structure of Chromosomes.

As 30-nm fibers, chromosomes are ~0.1 cm-long which is far bigger to what they are in the nucleus, thus, higher levels of folding are required. Although, in most cells interphase chromosomes are in a decondensed state that cannot be easily visualized, studies on amphibian lampbrush and insect polytene chromosomes show that decondensed, rather unfolded chromatin regions correlate with active transcription. In the lampbrush chromosomes these form elongated chromosome loops that emanate from a central, condensed chromatin core. In polytene chromosomes, they form distinct chromosome puffs between more condensed regions. Likewise, limited digestion of vertebrate chromatin with DNAse I shows that transcriptionally active or soon-to-be-active regions are digested immediately after the nuclease -hypersensitive sites (see above) but well before other more condensed chromatin. Two types of chromatin in eucaryotic cells: heterochromatin, which is highly condensed, makes about 10% of genome and is transcriptionally inactive, and euchromatin, which has several degrees of condensation and transcriptional activity. Chromatin of mitotic chromosomes is in its most condensed state which makes them visible with the light microscope. Each mitotic chromosome has a unique set of "bands" that result from its differential staining with fluorescent dyes. (Collage Figs 8-16,-17,-23,-25-30).

5. Chromosome Replication.
• Replication origins. Specific sequences that share an 11-bp core, identified in yeast by their ability to permit extrachromosomal replication to a plasmid. Initially named autonomously replicating sequences (ARS) (Fig 8-33). Similar sequences are thought to be present in other eucaryotic cells, although yet to be shown.
• Studies on a mammalian cell-free system using the SV 40 viral genome have shown that DNA replication in mammals is very similar in its enzymology to that in bacteria (Lecture 1). However, two important differences are worth mentioning: two DNA polymerases are needed: DNA polymerase a on the lagging strand and DNA polymerase d on the leading strand. Second, the mammalian DNA primase is itself a subunit of the DNA polymerase a and not part of the helicase as in bacteria. All the proteins but the T-antigen in this in vitro system come from the mammalian host. The T-antigen recognizes the viral ARS and utilizes ATP to induce the formation of the replication fork. Two replication forks that move in opposite direction form the replication bubble. The mammalian equivalent of the T-antigen is unknown (Fig 8-34).
• Pulse-chase experiments have shown that: (1) Replication origins in higher eucaryotes are activated in clusters (replication units) of 20-80 origins. (2) Replication units are activated at different times but reproducibly. (3)Individual origins are spaced 30000-300000 bp apart. (4)end of replication: when two replication forks moving in opposite direction collide head on or a chromosome end is reached.
• Highly condensed chromatin replicates late, while genes in active chromatin replicate early. Thus, transcriptionally active regions are more accessible to the replication machinery.
• Histone synthesis is tighly coupled to DNA replication. They are made during the S phase and are quickly degraded after DNA synthesis stops.
• Telomerase: the "end-replication problem" is solved by telomerase which is a special kind of Reverse Transcriptase that carries itself an RNA primer, complementary to the repeated telomere sequence (Fig 8-41).
6. RNA synthesis and processing.
• Three kinds of polymerases in eucaryotes: RNA polymerases I, II and III. Ià large rRNAs; IIà mRNAs and snRNAs; IIIà small, stable RNAs, i.e. 5S rRNA and tRNAs. RNA polymerases bind to specific regions known as promoters. RNA pol II produce transcripts that, in the nucleus are ~7000 nucleotides in average which is much more of the ~1200 nucleotides needed to code for average protein à introns + other non-coding regions. Elongation rate is constant for all genes: ~30 nucleotides/s while initiation rate varies a lot. Transcription initiation is a major control point for regulation of gene expression and will be studied in more detail in the next lecture.
• Primary transcripts or heterogeneous nuclear RNA (hnRNA): first mRNA transcripts in the nucleus. These transcripts are covalently modified at the 5í- and 3í-ends in the nucleus.
• 5í-end cap: addition of a methylated G nucleotide to the 5í first nucleotide (Fig 8-48). Capping occurs after only ~30 nucleotide of RNA have been synthesized. Functions: as we saw in lecture 1, the 5í cap plays an important role in translation initiation; it may also protect the growing RNA chain from degradation.
• 3í-poly A⁺ tail: In eucariotes there is no specific signal for transcription termination. Instead the growing transcript is cleaved at a specific site, 10 to 30 bp downstream of a AAUAAA sequence, and a tail of 100-200 A residues is added by a distinct poly-A polymerase to the cut 3í end. This completes the primary transcript, though, the RNA pol II may continue fruitlessly transcribing the downstream DNA (Fig 8-49). Functions: it aids in export of RNA from nucleus, it may affect stability of some mRNAs, and it also may tell the ribosome that the mRNA is intact and thus suitable for translatioon. Only RNA transcripts made by RNA pol II have 5í caps and 3í-polyA⁺ tails, i.e. protein factors needed bind to RNA pol II.
• RNA splicing (or intron removal). One of the student presentation will deal with the discovery of introns. Consensus acceptor and donor sequences for RNA splicing (Fig 8-53). Carried out by the spliceosome, a big complex of proteins and various small nuclear RNAs (snRNAs). In vitro studies suggest a two-step mechanism (Figs 8-54,-55). U2, U5 and U6 snRNAs have been identified as catalytic components of the spliceosome (Sontheimer and Steiz, 1993. Science, 262: 1989-1996). Thus, nuclear mRNA splicing may be an RNA-catalyzed process. Most of introns are spliced in sequence so that exons are joined togeher in frame. However, sometimes and through the action of other proteins on the spliceosme, the immediate 3í exon is skipped and either not spliced in or substituted by an alternative exon. This process of alternative splicing is a major mechanism for generating multiple protein products, with different functional properties, from a single gene and will be study in more detail in the next lecture.
• RNA-catalyzed splicing: self-splicing RNA introns in Tetrahymena (Fig 8-58), represent vestiges of very ancient mechanisms.
• mRNA export to the cytoplasm will be study in more detail later in the course.
• rRNA processing, (Fig 8-62). There are multiple rRNA coding genes in the genome around which a specialized structure called the nucleolus is formed. It is here where rRNA transcription and assembly of the ribosomes takes place.
• tRNA processing.