Biology Unit 3: The Nucleus

Unit 3: The Nucleus

• The nucleus serves as a storehouse for genetic information
• DNA replication occurs at the genomic level
• RNA transcription and processing are regulated
• Regulates gene expression, by controlling the transport of transcription factors from the cytoplasm to the nucleus.

Index

• The cell nucleus and the DNA
• Nuclear envelope
• DNA replication
• DNA transcription
• Traffic between the nucleus and the cytoplasm
• Nuclear bodies

3.1 The cell nucleus and the DNA

• Functions: serves as a storehouse for genetic information
• At the genomic level: DNA replication takes place
• RNA transcription and processing
• Regulates gene expression, by controlling the transport of transcription factors from the cytoplasm to the nucleus.

Chromosomes and chromatin

• Eukaryotic genomes are more complex than prokaryotic genomes, organized into multiple linear chromosomes. Instead of a single DNA molecule, the DNA is organized into multiple linear molecules
• DNA is organized into smaller packages (chromatin).
• Human DNA is 2 meters long but fits into a 5-10 micrometer nucleus
• DNA binds tightly to small proteins (histones) to form chromatin
• Chromatin exists in two forms:
• Chromatin: unwound and active in RNA synthesis
• Chromosomes: tightly wound and found during cell division and inactive for RNA synthesis

Heterochromatin and euchromatin

• Heterochromatin: condensed chromatin structure, inactive for transcription
• Euchromatin: loose chromatin structure, active for transcription
• Euchromatin is distributed throughout the nucleus, while heterochromatin is located around the periphery of the nucleus.

Chromatin structure

• Chromatin is a complex structure of DNA and proteins whose degree of condensation can vary depending on the cell cycle and chromosomal activity
• Levels of DNA packaging:
• 2nm fiber (double-stranded DNA)
• 11nm fiber ("beads-on-a-string" structure)
• 30nm fiber (solenoid structure)
• 300nm fiber (chromatin loops)
• 700nm fiber (condensed sections)
• 1400nm fiber (entire mitotic chromosome).

Histone modifications and DNA

• Histones have an amino-terminal tail that can be modified (acetylated, methylated, phosphorylated).
• These modifications constitute a "histone code" that influences DNA replication or expression
• Histone acetylation is associated with transcriptional activity
• Methylation can be associated with both active and repressed chromatin.

Chromosomes

• Each eukaryotic species has a characteristic chromosome number (e.g., humans have 46 chromosomes).
• Most cells are diploid (2n), having two sets of chromosomes (one from each parent).
• Gametes (sex cells) are haploid (n), having one set of chromosomes.
• 22 pairs of chromosomes are homologous (autosomes)
• Two sex chromosomes are either homologous or heterologous (XX for females and XY for males)
• Chromosomes have a centromere, dividing the chromosome into two arms (p-arm and q-arm).
• The ends of the chromatids are called telomeres
• Telomeres maintain structural integrity, position, ensure complete DNA replication of the chromosomes.

Telomerase

• Telomerase is a type of DNA polymerase involved in telomere formation
• It replicates telomeric DNA
• Telomeric DNA forms a loop structure protected by the protein complex shelterin
• Telomeres have specific sequences (TTAGGG in humans).

Genes and genomes

• Genome: the complete set of genetic material in an organism
• Gene: a segment of DNA that codes for a gene product (typically a protein).
• Contains structural and regulatory DNA
• Extragenic DNA (non-coding DNA) has various functions including regulation of gene expression.

DNA quantity paradox

• The amount of DNA in an organism does not directly correlate with the complexity or number of genes present in that organism.
• More complex organisms do not have more DNA than prokaryotes, due to a large percentage of repetitive DNA.

Gene structure

• Genes consist of exons (coding sequences) and introns (non-coding sequences).
• Introns are removed during RNA processing (splicing).
• Exons are spliced together to form mature mRNA.
• Alternative splicing allows a single gene to produce multiple protein isoforms.

Alternative splicing

• Alternative splicing generates a variety of mRNA transcripts from a single gene, which causes diversity in the proteins an organism can produce
• This results in many more different proteins than expected based on the number of genes.

Complexity in human DNA

• Human genomes contain 21000 genes, but also non-coding DNA (regulatory sequences, repetitive sequences).
• Regulatory sequences: promoters, silencers, enhancers
• Repetitive DNA sequences: tandem repeats (satellite DNA), sparse repeats (LINES, SINES, LTR, transposons), gene duplication and pseudogenes
• The human genome contains more non-coding sequence than coding sequence

Non-coding RNA

• ncRNA: RNA molecules that do not encode proteins. Examples include tRNA, rRNA, miRNA and long ncRNA (lncRNA).
• miRNAs: small non-coding RNAs that regulate gene expression via RNA interference
• IncRNAs: long non-coding RNA molecules involved in gene regulation.

RNA processing

• Bacterial mRNAs are used immediately for protein synthesis while still being transcribed
• Ribosomal, Transfer, and eukaryotic mRNAs must be processed
• 5' capping, 3' polyadenylation, splicing of introns are examples of RNA processing steps carried out on pre-mRNAs

3.2 Nuclear envelope

• Structure of the nuclear envelope
• Nuclear pore complex

Nuclear envelope

• It is a selective barrier that prevents the free traffic of molecules between the nucleus and the cytoplasm; only nuclear pore complexes allow communication between both compartments.
• Key role in regulation of gene expression, maintaining the internal composition of the nucleus, and keeping the two compartments metabolically independent.

Nuclear envelope structure

• 2 nuclear membranes: outer and inner membrane
• Nuclear lamina
• Nuclear pore complex

Outer nuclear membrane

• Connected to the endoplasmic reticulum membrane.
• Ribosomes are frequently attached to the outer membrane.
• Membrane proteins bind to the cytoskeleton.

Inner nuclear membrane

• Contains specific membrane proteins that bind the nuclear lamina.

Nuclear pore complex

• Junction of inner and outer membranes.
• The only channels that allow molecules and larger macromolecules to pass through.
• Involved in selective trafficking of proteins and RNA between the nucleus and cytoplasm.

Nuclear lamina

• Fiber network that provides structural support to the nucleus.
• Made up of proteins lamin A, B, and C.
• All lamins are fibrous proteins between 60-80 kDa.
• Lamins are joined together to form filaments.

3.3 DNA replication

• Semi-conservative mechanism: the two strands separate to form templates for producing new strands, thus producing an identical copy.
• DNA polymerases are essential enzymes; they catalyse the addition of dNTPs onto the 3'OH group of the growing strand, continuously in the 5' → 3' direction.
• Replication begins at origins of replication, where initiator proteins separate the DNA strands to form replication forks.
• Replication proceeds in both directions from the origin of replication, creating replication bubbles or forks
• Prokaryotes have a single origin, whereas eukaryotes have multiple.

DNA polymerase

• Various in eukaryotes and prokaryotes with specific roles in replication and repair
• Bacteria use DNA polymerase III mostly for replication
• Eukaryotes use α, δ, ε polymerases for nuclear DNA and γ for mitochondrial DNA.

Origin of replication

• DNA molecule opens like a zipper by breaking hydrogen bonds between complementary bases.
• Initiator proteins recognize specific nucleotide sequences (origins of replication) on DNA, and stimulate the separation of strands and connection of other proteins needed for replication.
• A large number of enzymes and proteins are involved in replication forming a complex called replisome
• Replication in prokaryotes starts at a single origin.
• Replication in eukaryotes begins at multiple origins.

Replication fork

• The place where the parental DNA strands separate, allowing new strands to be synthesized.
• Replication proceeds in both directions from the origin of replication.
• Leading strand is synthesized continuously
• Lagging strand is synthesized discontinuously in small fragments (Okazaki fragments).
• Enzmyes and proteins like DNA polymerase, helicase, primase, and ligase are involved.

DNA maintenance

• The accuracy of DNA replication is critical for cell reproduction.
• Error frequency is less than one incorrect base for every 10^9 incorporated nucleotides.
• Mechanisms by which DNA polymerase achieves high accuracy:
• Correct base selection.
• Double-reading capability (exonuclease activity in 3'→5' direction).

3.4 DNA transcription

• The process of synthesizing an RNA molecule from a DNA template.
• DNA strands have different functions in transcription
• The antisense strand acts as the template for RNA synthesis.
• The RNA transcript is complementary to the antisense strand and identical to the sense strand, except for uracil in place of thymine
• RNA polymerase is the main enzyme involved; it adds ribonucleotides to the 3' end of the growing RNA chain, in 5'→3' direction.

RNA polymerase

• RNA polymerase is the main enzyme involved in RNA synthesis
• Catalysed reaction: (NMP)n + NTP → (NMP)n+1 + PPi
• NMP: nucleotide monophosphate
• NTP: nucleotide triphosphate
• PPi: pyrophosphate

Initiation of transcription

• Transcription begins with the binding of RNA polymerase to the promoter
• The promoter region extends for tens or hundreds of bases before the transcription start site, containing consensus sequences that are repeated in different genes
• DNA unwinds and RNA polymerase undergoes conformational and chemical changes (phosphorylation) that induce the initiation of transcription

Elongation of transcription

• Most of the transcription factors are released at the beginning of the elongation.
• RNA polymerase moves in 5ʹ → 3ʹ direction by adding nucleotides to the 3′ end of the growing RNA molecule
• DNA strand that is being transcribed unwinds and rewinds during the polymerase movement, so that the DNA-RNA hybrid region remains relatively short.

Termination of transcription

• RNA polymerase recognizes certain DNA sequences that lie at the end of genes.
• This ends RNA synthesis, followed by the release of the transcription machinery

3.5 Traffic between the nucleus and the cytoplasm

• Selective transport of proteins to and from the nucleus
• Regulation of protein transport to the nucleus
• RNA Transport
• Proteins necessary for nuclear functions must enter the nucleus from synthesis sites in the cytoplasm
• Importins recognize the NLS ('Nuclear localization signal') of the cargo protein and transport it from the cytoplasm to the nucleus
• Ran proteins:
• Guanosine di/triphosphate binding protein
• High concentration of GTP in the nucleus determines directionality of nuclear transport
• Enzymes that stimulate the exchange of GDP for GTP on Ran - are located on the nuclear side of the envelope.
• Exportins: recognize NES ('Nuclear export signal') responsible for proteins leaving the nucleus.
• Regulation of transport: some cytoplasmic proteins mask the NLS, preventing proteins from entering the nucleus; proteins also regulated by phosphorylation

3.6 Nuclear bodies

• Differentiated organelles in the nucleus
• Compartmentalize the nucleus for RNA and protein concentration that functions in certain processes
• Dynamic structures without membranes, maintain integrity via interactions with proteins and RNA.

Nuclear bodies summary

• Nucleolus: rRNA transcription, processing, ribosome assembly
• Cajal body: snRNP assembly, modification of snRNAs
• Clastosome: proteasomal activity, histone pre-mRNA processing
• Histone locus body: storage of histone pre-mRNAs
• Speckle: transcriptional regulation, DNA repair
• PML body: transcriptional regulation, DNA repair
• Polycomb body: gene silencing

Explore the intricate functions of the cell nucleus in this quiz. Learn about its role as a storehouse for genetic information, the processes of DNA replication and RNA transcription, and the regulation of gene expression. Test your understanding of nuclear components and their significance in eukaryotic organisms.