Molecular Biology: DNA Replication Quiz

Unit 3: The Nucleus

• The nucleus serves as a storehouse for genetic information
• DNA replication takes place at the genomic level
• RNA transcription and processing occur
• Gene expression is regulated by controlling the transport of transcription factors from the cytoplasm to the nucleus

Index

• 3.1: The cell nucleus and the DNA
• 3.2: Nuclear envelope
• 3.3: DNA replication
• 3.4: DNA transcription
• 3.5: Traffic between the nucleus and the cytoplasm
• 3.6: Nuclear bodies

3.1 The cell nucleus and the DNA

• Functions:
  • Stores genetic information
  • Site of DNA replication
  • Site of RNA transcription and processing
  • Regulates gene expression by controlling the transport of transcription factors

Chromosomes and chromatin

• Eukaryotic genomes are complex, organized on multiple linear chromosomes
• DNA is packaged with small proteins called histones
• The length of human DNA is 2 meters, but it must fit in a core of 5-10 micrometers within the nucleus

Chromosomes

• Tightly packaged DNA
• Found only during cell division
• DNA is not used for macromolecule synthesis

Chromatin

• Unwound DNA
• Found throughout interphase
• DNA is being used for macromolecule synthesis

Heterochromatin and euchromatin

• Heterochromatin: condensed chromatin, inactive for transcription
• Euchromatin: loose chromatin, active for transcription

Levels of DNA packaging

• Level 1: DNA double helix (2nm) coils around histone core to form nucleosome ("beads-on-a-string")
• Level 2: Nucleosomes coil to form 30nm chromatin fiber
• Level 3: 30nm fibers form loops attached to a protein scaffold, forming a 300nm fiber
• Level 4: 300nm fibers condense forming a 700nm fiber or chromatid which is further condensed

DNA polymerase

• Eukaryotes and prokaryotes contain DNA polymerases with different roles
• Bacterial DNA polymerase III is the main polymerase responsible for replication
• In eukaryotes, DNA polymerases α, δ and ε function in nuclear DNA replication, and DNA polymerase γ is for mitochondrial DNA replication
• DNA polymerases synthesize DNA 5' to 3'

Origin of Replication

• DNA molecule unwinds at specific points called origins of replication
• Initiator proteins recognize specific nucleotide sequences to facilitate the attachment of other proteins to form two replication forks
• A large replication complex (replisome) is assembled consisting of multiple enzymes and proteins

Origin of Replication in Prokaryotes

• Replication begins at one single origin
• Initiator protein unwinds the DNA, recruiting other replication enzymes
• Replication proceeds in both directions from the single origin

Origin of Replication in Eukaryotes

• Eukaryotes require multiple origins of replication to replicate long chromosomes in a reasonable time
• The rate of DNA replication is slower in eukaryotes than in bacteria due to DNA packaging in chromatin

Replication Fork

• The DNA molecule unwinds to separate parental strands forming two replication forks.
• One strand is synthesized continuously (leading strand) whereas the other in short fragments (lagging strand)

DNA Maintenance

• High accuracy in DNA replication is crucial for cell reproduction
• Error frequency is less than one incorrect base per 10,000 nucleotides
• Mechanisms for DNA polymerase fidelity include:
  • Selecting correct bases
  • Proofreading activity (3' to 5' exonuclease excises incorrect base)

Telomerase

• DNA polymerase involved in telomere formation
• Contains 159-nucleotide RNA oligonucleotide that acts as a template for telomere synthesis
• Replicates telomeric DNA

Genes and Genomes

• Genome: The complete set of genetic material in an organism
• Gene: A segment of DNA that codes for a specific gene product, either RNA or protein
• Extragenic DNA: DNA in a genome that does not contain protein-coding genes, but can have regulatory functions

DNA Quantity Paradox

• A larger genome does not necessarily correspond to a greater number of genes
• Complexity is a multi-faceted parameter and not only a matter of DNA quantity

Gene Structure: Introns and Exons

• Exons: Regions of mRNA that are expressed and form protein-coding sequences
• Introns: Non-coding regions of DNA that are removed during mRNA processing

Alternative Splicing

• Multiple mature mRNA transcripts can be generated from a single gene depending on which exons are included or excluded in the final mRNA
• Increases the diversity of proteins encoded by a single gene and plays a critical role in development and differentiation.

Complexity in Human DNA: Types of Sequences in our genome

• Regulatory DNA sequences include promoters, silencers, and enhancers
• Extragenic DNA includes repetitive sequences (tandem and sparse repeats), gene duplication, and pseudogenes

Noncoding RNA

• Diverse classes of noncoding RNAs play important regulatory roles in the cell (tRNAs, rRNAs, miRNAs, IncRNAs)
• miRNA: 20-30 nucleotides, control gene expression by inhibiting translation
• IncRNA: over 200 nucleotides, involved in regulating gene expression and chromatin structure

RNA Processing

• Bacterial mRNAs are used immediately for protein synthesis
• Many types of RNA (rRNA, tRNA, mRNA) in eukaryotes are processed extensively before being used to synthesize proteins.

Ribosomal RNAs

• rRNA molecules of both prokaryotes and eukaryotes are derived from a single long pre-rRNA molecule (cleaved into smaller mature rRNA)
• Each rRNA type (18S, 5.8S, 28S, 5S) is transcribed from a separate gene (except in the case of the 5.8S, 18S and 285 rRNAs, which are transcribed from a single long pre-rRNA molecule) in eukaryotes

tRNAs

• tRNA molecules are processed from longer precursors, requiring cleavage of both 5' and 3' ends
• Modification of specific bases in precursor tRNA (10%)

mRNA Processing (Eukaryotes)

• 5' cap
• 3' poly-A tail
• Splicing: removal of introns

Splicing

• Splicing proceeds in two steps:
  1. Cleavage and joining
  2. Excising intron loop
• Alternative splicing: multiple mature mRNAs from one gene

Nuclear Envelope and Pore Complex

• Double membrane structure surrounding the nucleus
• Nuclear pore complex is the pathway for selective transport between the nucleus and cytoplasm
• The nuclear lamina is a fibrous network that provides structural support to the nucleus

Selective Transport of Proteins

• Proteins required for nuclear functions are transported from the cytoplasm to the nucleus
• NLS (nuclear localization signals): specific amino acid sequences targeting particular proteins to the nucleus
• Importins: bind to NLSs, facilitating transport
• Ran proteins: regulate the directionality of transport

RNA Transport

• RNA molecules are transported through the nuclear pore complex, associated with proteins to form RNPs
• Different RNA types (tRNA, mRNA, rRNA, miRNA) use various mechanisms

Other Small Noncoding RNAs

• snRNAs (small nuclear RNAs): act in pre-mRNA splicing
• snoRNAs (small nucleolar RNAs): involved in rRNA processing

Nucleolos

• Large structure in the nucleus
• Site of ribosome biogenesis

Histone Modifications

• Acetylation: adding acetyl groups, relaxing chromatin, increasing transcriptional activity
• Methylation: adding methyl groups, affecting transcriptional activity

Chromatin Remodeling Factors

• Protein complexes that alter interactions between DNA and histones, impacting transcription

DNA Methylation

• Epigenetic mechanism for controlling transcription (often leads to repression)
• Methylation of cytosine bases preceding guanines (CpG dinucleotides)

Regulation of Transcription

• Mechanisms for regulating gene expression in eukaryotes by:
  • Binding of regulatory proteins
  • Chromatin structure modifications
  • DNA methylation
  • Noncoding RNA regulation

Nuclear Bodies

• Specific subnuclear structures that concentrate specific proteins and RNAs
• Include the nucleolus, cajal bodies, speckles, histone locus bodies, etc

Test your knowledge on the key processes in DNA replication, focusing on eukaryotic systems. Explore the roles of DNA polymerases, initiator proteins, and the significance of origins of replication. This quiz also delves into the functions of miRNAs and the structural characteristics of nuclear components.