DNA Replication, Transcription, and Translation in Eukaryotes

DNA Replication

Overview of DNA Replication

DNA replication is the process by which a cell duplicates its DNA, ensuring that each daughter cell receives an identical copy. This process is fundamental to cell division and inheritance.

• Strand separation occurs at regions called replication forks.

• Replication begins at one or more fixed sites known as origins of replication.

• Replication is semiconservative: each daughter duplex contains one parental and one newly synthesized strand.

• Bidirectional replication means that replication proceeds outward in both directions from each origin.

Eukaryotic Replication of a Linear Chromosome

Eukaryotic chromosomes are linear and contain multiple origins of replication to ensure timely duplication of large genomes.

• Replication is bidirectional and proceeds from several fixed origins.

• Replication forks advance until they meet another fork traveling in the opposite direction.

• Origins are programmed to initiate replication at fixed times during the S phase of the cell cycle.

DNA Polymerases: Enzymes Catalyzing Chain Elongation

DNA polymerases are the enzymes responsible for synthesizing new DNA strands by adding nucleotides to a primer strand.

• DNA polymerase catalyzes the nucleophilic attack by the 3'-OH group at the primer terminus upon the α-phosphate of the incoming deoxynucleoside triphosphate (dNTP) that is base-paired with the template.

• This reaction forms a new phosphodiester bond and releases pyrophosphate.

Equation:

$(dNMP)_n + dNTP \rightarrow (dNMP)_{n+1} + PP_i$

• DNA polymerase requires a DNA template, an RNA or DNA primer, and dNTPs.

• DNA polymerase I has both 3'→5' exonuclease (proofreading) and 5'→3' exonuclease (primer removal) activities.

Discontinuous Synthesis on the Lagging Strand

Because DNA polymerases can only synthesize DNA in the 5'→3' direction, replication of the lagging strand is discontinuous.

• The leading strand is synthesized continuously toward the replication fork.

• The lagging strand is synthesized discontinuously in short fragments called Okazaki fragments.

• Each Okazaki fragment requires a new RNA primer.

Nick Translation and Removal of RNA Primers

RNA primers must be removed and replaced with DNA to complete replication.

• Primase synthesizes short RNA primers to initiate DNA synthesis.

• DNA polymerase I removes RNA primers (5'→3' exonuclease activity) and replaces them with deoxynucleotides.

Schematic View of the Replication Fork

The replication fork is a complex structure where multiple proteins coordinate the unwinding and synthesis of DNA.

• Key components include: DNA polymerase, primase, helicase, single-stranded DNA-binding proteins (SSB), and topoisomerase.

• The sliding clamp (β subunit) increases the processivity of DNA polymerase.

Helicase-Mediated DNA Unwinding

Helicase is essential for unwinding the double helix, allowing replication machinery access to single-stranded DNA.

• Helicase catalyzes the ATP-dependent unwinding of double-stranded DNA.

• Unwinding creates torsional stress, which is relieved by topoisomerase.

Transcription in Eukaryotic Cells

RNA Polymerases and Transcription Factors

Eukaryotic transcription involves three distinct RNA polymerases, each responsible for synthesizing different classes of RNA.

• RNA polymerase I (pol I): transcribes major ribosomal RNA genes.

• RNA polymerase II (pol II): transcribes protein-coding genes and some small RNA genes.

• RNA polymerase III (pol III): transcribes small RNA genes (e.g., tRNA, 5S rRNA).

• Transcription factors (TFI, TFII, TFIII) are required for initiation by each polymerase.

TFIIIA and Zinc Finger Proteins

Zinc finger proteins are a common motif in transcription factors, allowing them to bind specific DNA sequences.

• The α-helices of zinc finger proteins fit within the major grooves of DNA.

• These proteins are modular and can be strung together to recognize extended DNA sequences.

Structures of Eukaryotic Promoters

Promoters are DNA sequences that define where transcription of a gene by RNA polymerase begins.

• The TATA box is a core promoter element, analogous to the bacterial -10 region.

• Additional regulatory sites, called enhancer regions, may exist several kilobase pairs upstream from the initiation site.

DNA Looping and Transcription Activation

DNA looping allows activator proteins bound to enhancers to interact with the core transcription machinery at the promoter.

• Pol II interacts with several transcription factors, including TATA box-binding protein (TBP) and TFIIA, -B, -E, -F, and -H.

• TBP binds to DNA and bends it by 90°, facilitating assembly of the transcription complex.

Histone Acetylation and Transcriptional Activity

Histone acetylation is a key epigenetic modification that regulates gene expression.

• Acetylation of core histones neutralizes basic lysine residues, weakening ionic interactions between histones and DNA.

• High levels of histone acetylation are associated with increased transcriptional activity.

Termination of Transcription in Eukaryotes

Termination of transcription by RNA polymerase II involves cleavage of the pre-mRNA and addition of a poly(A) tail.

• RNA polymerase II transcribes past the end of the gene, passing through one or more TTATT signals.

• The pre-mRNA is cleaved 11 to 30 nucleotides downstream of the AAUAAA signal.

• A poly(A) tail is added by poly(A) polymerase, which enhances mRNA stability (the longer the tail, the longer the half-life).

Posttranscriptional Processing

5'-Capping of Pre-mRNA

Eukaryotic pre-mRNA is modified at the 5' end by the addition of a 7-methylguanosine cap.

• The cap structure protects mRNA from degradation and is involved in translation initiation.

Splicing of Pre-mRNA

Splicing removes non-coding sequences (introns) from pre-mRNA, joining together the coding sequences (exons).

• Small nuclear ribonucleoproteins (snRNPs), such as U1 and U2, aid in pre-mRNA loop formation.

• The snRNP-pre-mRNA complex is called the spliceosome.

Alternative Splicing

Alternative splicing allows a single gene to produce multiple protein isoforms by varying the combination of exons included in the final mRNA.

• Example: The α-tropomyosin gene in rats can generate seven different mRNAs through alternative splicing pathways.

Translation: Protein Synthesis

Overview of Translation

Translation is the process by which the genetic code carried by mRNA is decoded to produce a specific polypeptide.

• The ribosome uses an mRNA template and aminoacyl-tRNAs to synthesize a polypeptide.

• mRNA is read 5'→3', and the polypeptide is synthesized from the N-terminus to the C-terminus.

Activation of Amino Acids

Amino acids are activated for incorporation into proteins by attachment to tRNA molecules.

• The anticodon loop of tRNA contains a trinucleotide sequence complementary to the codon in mRNA.

• Aminoacyl-tRNA synthetase (aaRS) catalyzes the attachment of amino acids to their corresponding tRNAs in two steps:

  1. The amino acid is activated by ATP to form aminoacyl adenylate.

  2. The activated amino acid is transferred to the tRNA, releasing AMP.

The Genetic Code

The genetic code is a set of rules by which information encoded in mRNA is translated into proteins.

• The AUG start codon encodes methionine and is used as the initiation codon in eukaryotes.

• There are three stop codons (UAA, UGA, UAG), also called nonsense codons, which signal termination of translation.

Major Participants in Translation

Translation requires mRNA, tRNA, and ribosomes.

• The ribosome is a large ribonucleoprotein complex composed of about 60–70% RNA and 30–40% protein.

• Bacterial ribosomes are 70S (50S and 30S subunits); eukaryotic ribosomes are 80S (60S and 40S subunits).

• tRNAs occupy three sites in the ribosome: A (aminoacyl), P (peptidyl), and E (exit).

Mechanism of Translation

Translation occurs in three main stages: initiation, elongation, and termination.

Stage 1 – Initiation

• Initiation factors (IF1, IF3) facilitate dissociation of the 70S ribosome in bacteria.

• mRNA and initiator tRNA bind the 30S subunit with the help of IF2.

• The 50S subunit binds to the 30S initiation complex, aligning the initiator tRNA in the P site.

Stage 2 – Elongation

• The peptide chain is attached to the tRNA in the P site; the A and E sites are empty at the start of each cycle.

• Elongation factors (e.g., EF-Tu) help position the correct aminoacyl-tRNA in the A site.

• Peptide bond formation is catalyzed by the peptidyl transferase activity of the ribosome.

• The ribosome translocates, moving the peptidyl-tRNA to the P site and freeing the A site for the next aminoacyl-tRNA.

Stage 3 – Termination

• When a stop codon enters the A site, release factors (RF1 for UAA, RF2 for UAG and UGA) bind, promoting hydrolysis of the peptidyl-tRNA bond.

• The ribosome dissociates from the mRNA, and the subunits are recycled for another round of translation.

DNA Methylation, Gene Silencing, and Epigenetics

DNA Methylation in Eukaryotes

DNA methylation is an epigenetic modification that plays a critical role in gene regulation, development, and disease.

• Methylation patterns are altered in cancer and are important for understanding carcinogenesis.

• Methylated cytosines (Cs) are typically found upstream from guanines (Gs) in CpG dinucleotides.

• CpG regions are underrepresented in eukaryotic genomes, but CpG islands (regions of expected CpG frequency) are found near gene promoters.

• DNA methylation patterns are heritable and maintained by DNA methyltransferases (DNMTs): DNMT1 (maintenance), DNMT3a and DNMT3b (de novo methylation).

• DNA methylation can lead to permanent gene inactivation; demethylation can activate repressed genes.

• DNA CpG methylation is responsible for X chromosome inactivation and gene imprinting.

• In females, one X chromosome is inactivated by methylation; in imprinting, only one parental allele is expressed while the other is silenced by methylation.