Internal

Internal Organization of the Nucleus and Ribosome Biosynthesis

• The nucleus has a double membrane nuclear envelope
• The nuclear envelope has pores (0.25 µm surface area) for ribosome passage
• There are ribosome pore complexes (1 µm) on the nuclear envelope and nuclear lamina (TEM).
• The rough endoplasmic reticulum(ER) is connected to the nuclear envelope
• The structure of ribosome pore complex is visible in Transmission Electron Microscopy (TEM) images.

Nucleolus

• The nucleolus is the largest structure in the nucleus of eukaryotic cells

• It is 1 μm in diameter

• It is stained with basic dyes

• It can be difficult to observe if nuclear chromatin is very condensed

• The nucleolus is composed of proteins + RNA (1940)

• It is the site of ribosomal RNA production (1960)

• RNA transcription, processing, and ribosome assembly take place within the nucleolus

• Actively growing cells contain 5-10 million ribosomes

• Nucleoli are well developed in cells active in protein synthesis. This is especially true during rapid cell growth (like in young cells, cells of the pancreas, and various cancers)

Nucleolus Contains

• rRNA genes (DNA regions extending into the nucleolus)
• Pre-rRNA transcripts
• Mature rRNA transcripts
• Pre-ribosomal subunits

Nucleolus Organization

• The nucleolus is organized around specific chromosomal regions (13, 14, 15, 21, 22)
• These chromosomes contain the genes for 5.8S, 18S, and 28S rRNAs.
• Cells contain repeated rRNA genes (multiple copy genes).
• The rRNA genes, including repeats and the resulting ribosomal RNA molecules, are crucial for ribosome function.

Ribosomes

• Ribosomes are the sites of protein synthesis in both prokaryotic and eukaryotic cells

• Prokaryotic and eukaryotic ribosomes are composed of two distinct subunits (each with specific proteins and rRNA components.

• E. coli ribosomes account for 25% of the dry weight of the cell.

• Ribosomes are found in mitochondria and chloroplasts (of eukaryotes). Mitochondria and chloroplast ribosomes resemble bacterial ribosomes

• Ribosomes have a function in protein production

• Ribosomes are composed of rRNA + proteins

Types of Ribosomes

• Free Ribosomes:

  • Located in the cytosol
  • Occur in greater numbers in cells that retain most of their manufactured protein
  • For proteins that stay in the cytoplasm or form cytoplasmic structural or motile elements

• Bound Ribosomes:

  • Attached to the endoplasmic reticulum (ER)
  • Occur in greater numbers in cells that secrete their produced proteins (like in pancreatic cells).
  • For proteins packaged into vesicles (for storage/export) or form membranes

Functions of Ribosomes

• Assemble amino acids into proteins (proteins are necessary for cellular functions)
• The process of protein production proceeds via DNA transcription and mRNA translation
• The genetic message from mRNA is translated into proteins during DNA translation
• Protein assembly sequences are in the mRNA
• mRNA is synthesized in the nucleus and exported to the cytoplasm for protein synthesis
• Ribosomes located in the cytoplasm complete protein production.

Bacterial Ribosomes

• The general structure of prokaryotic and eukaryotic ribosomes are similar.
• E. coli ribosome small subunit (30S), consists of 16S rRNA and 21 proteins
• E. coli ribosome large subunit (50S), consists 23S rRNA and 5S rRNA and 34 proteins.
• A ribosome is composed of large and small subunits; each comprises proteins + rRNA molecules.

Eukaryotic Ribosomes

• Eukaryotic ribosomes' subunits are larger and have more proteins than prokaryotic ones
• The small subunit (40S) of eukaryotic ribosomes consists of 18S rRNA and ~30 proteins.
• The large subunit (60S) contains 28S, 5.8S, 5S rRNAs, and 45 proteins.
• Proteins and RNA make up the subunits.

5S rRNA

• 5S rRNA is not located in the nucleolus
• There are ~2000 copies of the gene that encodes 5S rRNA
• 5S rRNA genes are present in a single cluster on chromosome 1.
• 5S rRNA transcription takes place outside the nucleolus (done by RNA polymerase III).

Transcription & Processing of rRNAs

• Transcription of large 45S pre-rRNA molecules (includes 18S, 5.8S, 28S rRNAs)
• Processing involves
  • Cleavages to make precursors for the large and small ribosomal subunits
  • Chemical modifications
• Pre-rRNA is modified to create the individual rRNAs that go on to combine with proteins to create the ribosome components.

Chemical Modifications and Nucleolytic Processing

• pre-rRNA is chemically modified (involving RNA molecules and proteins)
• pre-rRNA is processed through nucleolytic digestion, creating 18S, 5.8S and 28S rRNA molecules.
• There are ~300 proteins involved.

Other Nucleolus Functions

• Site of other RNA production
• Site of other RNA-protein complex assembly
• U6 SnRNP (small nuclear ribonucleoprotein) part of mRNA splicing processes is an example.

Chromatin

• Chromatin is the packaging material for DNA

• Chromatin is composed of DNA + proteins (histones and non-histone proteins)

• Chromatin structure is important for cell division, preventing tangles and damage to DNA during division

• Basic dyes bind to it; the Feulgen reaction is specific for identifying DNA

Euchromatin

• Lightly stained (invisible with light microscopy; visible with electron microscopy)
• Less condensed; uncoiled and dispersed form of chromatin
• Active chromatin; actively transcribed sequences
• Contains many of the genes that are actively transcribed in a cell

Heterochromatin

• Dark staining

• Dense, tightly packed form

• In the nuclear periphery

• Contains inactive, condensed DNA

• Located at centromeres and telomeres

• More than 10% of the mammalian genome is packaged into heterochromatin

• The cell must condense and de-condense chromatin for DNA transcription

Fine Structure of Chromatin

• Basic structural unit = nucleosome

• Roger Kornberg described the nucleosome in 1974

• Chromatin spreads over a lower salt concentration solution to study via transmission electron microscopy (EM)

• A 10 nm chromatin fiber has a beaded appearance

• Nucleosome = a disc-shaped structure

• DNA is coiled around the core of an octamer of histone molecules.

• 5 major types of histones exist

  • H2A, H2B, H3, and H4 (nucleosomal)
  • H1 (linker)

• Extended DNA length = 2 meters for a human somatic cell

• The DNA needs to fit in a cell nucleus = only 5-10 µm in diameter

• Histones create order and tightly wrap DNA within the cell nucleus

• A histone octamer is formed via 2 copies of H2A, H2B, H3, and H4

• DNA winds around the octamer core (approximately 146 bp and ~1.75 turns)

• DNA connecting two nucleosomes = Linker DNA (~80 bp)

• Histone H1 attaches to the Linker DNA, changing conformation.

• Chromatin's further condensation is achieved through 30 nm chromatin fiber formation

• 10 nm chromatin fiber folds into a thicker filament = 30 nm chromatin fibre

• Interactions between H1 histones are important for 30 nm chromatin condensation structure

• The 30 nm fibers coil into a more tightly condensed form.

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