UWorld Molecular Biology

• DNA polymerase is used to replicate DNA, while RNA polymerase transcribes DNA into RNA.
• DNA polymerase reads DNA in the 3′ → 5′ direction but synthesizes DNA in the 5′ → 3′ direction.
• DNA polymerase links the 3′ OH of the growing strand to the 5′ phosphate of incoming nucleotides.
• Each round of DNA replication reduces the percentage of helices that contain original (15N) strands, illustrating semiconservative replication.
• Double-strand breaks (DSBs) occur in the deoxyribose-phosphate backbone of DNA, and their repair requires the formation of phosphodiester bonds.
• Mismatch repair (MMR) fixes errors made during DNA replication by replacing mismatched DNA nucleotides, but it does not act on RNA.
• DNA synthesis is a condensation reaction that releases water.
• DNA synthesis is exergonic and releases energy through pyrophosphate hydrolysis.
• Activators increase transcription by helping RNA polymerase bind to DNA, but repressors inhibit transcription by blocking RNA polymerase.
• RNA polymerase is essential for transcription, but not for DNA replication
• To interpret RT-PCR results, reverse complement the cDNA sequence to find the original mRNA sequence, replacing thymine (T) with uracil (U).
• Alternative splicing allows one gene to produce multiple proteins but does not cause codon redundancy.
• The poly-A tail (at the 3′ end of mRNA) protects mRNA and facilitates nuclear export, while the 5′ cap prevents degradation and aids in ribosome binding.
• The genetic code is redundant (degenerate), meaning multiple codons can code for the same amino acid, but it is not ambiguous because each tRNA carries only one amino acid.
• Wobble base pairing allows for variation in the third codon position without changing the encoded amino acid.
• Types of mutations include: silent (no amino acid change), missense (amino acid substitution), nonsense (premature stop codon), and frameshift (insertion/deletion altering the reading frame).
• Translation forms peptide bonds through condensation reactions.
• Ribosomal RNA (rRNA) builds ribosomes and catalyzes peptide bond formation.
• Eukaryotic ribosomes are 80S (60S + 40S) and synthesize proteins in the cytoplasm or on the rough ER.
• Prokaryotic ribosomes are 70S (50S + 30S) and are not used in eukaryotic cells.
• Ribosomes attach to the rough ER for the synthesis of secretory proteins.
• Cap-dependent translation decreases with increasing competition from cap analogs, while IRES-mediated translation remains unaffected.
• MicroRNAs (miRNAs) silence genes by binding to complementary mRNA, which blocks translation or degrades the mRNA.
• Small nuclear RNAs (snRNAs) splice pre-mRNA in the nucleus and are not involved in translation or gene silencing.
• Complementary DNA (cDNA) lacks introns and does not require splicing, but it still produces the same protein as the gene it was derived from.
• cDNA, derived from mature mRNA, lacks introns and does not undergo splicing when expressed but still produces the same protein as the endogenous gene.
• To analyze mRNA half-life, convert mRNA to cDNA and track its degradation over time using qRT-PCR.
• Euchromatin (characterized by acetylated histones) is open and actively transcribed, while heterochromatin (deacetylated/methylated histones) is tightly packed and has low transcriptional activity.
• Histone acetylation loosens chromatin and increases transcription, while deacetylation condenses chromatin and decreases transcription.

Regulation of Transcription

• Increasing histone acetylation generally raises gene expression by opening DNA structure.
• Inhibiting enzymes that remove chemical groups (like acetyl groups) increases those modifications, typically boosting gene expression by loosening DNA structure.
• Acetylation opens chromatin structure (euchromatin), increasing DNA accessibility and boosting gene transcription.
• Deacetylation closes chromatin structure (heterochromatin), decreasing DNA accessibility and reducing gene transcription.
• Mutations in enhancers can increase transcription by allowing more transcription activators to bind, raising gene expression and potentially increasing disease risk.
• When repressors act, they inhibit transcription by preventing RNA polymerase from binding to the promoter.
• Conversely, activators facilitate transcription by helping RNA polymerase bind more efficiently.
• Antisense oligonucleotides (ASOs) bind to pre-mRNA and affect its splicing (processing), leading to changes in exon inclusion, increasing functional protein production, and reducing symptoms of diseases like SMA.

TERT

• In somatic cells, TERT (telomerase reverse transcriptase) is rarely expressed and stored in heterochromatin, limiting its access to RNA polymerase.
• In somatic cells, genes that are rarely transcribed, like TERT, are typically stored in heterochromatin, a tightly packed DNA form that limits accessibility to RNA polymerase and reduces gene expression.

Gels

• Full-length mRNA (all exons included) is the largest and moves the least in gel electrophoresis.
• Shorter or truncated mRNA (exon removed) moves further down.
• Small RNAs (snRNA) are the smallest and move the farthest down.
• In nonreducing SDS-PAGE, a homodimer (WT) moves slower due to intact disulfide bonds, while a monomer (mutant) moves faster due to the absence of disulfide bonds.
• Northern blotting separates mRNA by size, with smaller fragments migrating further down the gel.

Splicing

• Splicing occurs after transcription is complete and does not affect RNA synthesis.
• Splicing occurs before mature mRNA leaves the nucleus and does not control transport.
• Splicing does not directly alter translation but indirectly affects it by changing the mature mRNA sequence.
• snRNA/snRNPs edit (splice) pre-mRNA in the nucleus and are not involved in gene silencing or translation control.

DNA Modification

• DNA methylation occurs on cytosine (C) and reduces transcription.

Restriction Enzymes

• Restriction Enzymes recognize and cut palindromic sequences, but telomeres (TTAGGG) are not palindromes and aren't cut.

General Concepts

• Complementary nucleic acid strands hybridize, while proteins and antibodies do not.
• Labeled RNA probes can confirm gene expression by detecting complementary mRNA.
• Vector DNA construction uses reverse transcriptase, DNA polymerase, restriction enzymes, and ligase.
• Gene duplication creates similar genes that mutate over time and acquire different functions.
• Bacterial DNA can be transferred through conjugation (direct DNA transfer) or transformation (uptake of foreign DNA from the environment).

Proteins

• Hyperphosphorylated proteins (e.g., in Alzheimer's disease) require increased phosphatase activity or decreased kinase activity to restore function.
• Ribosome assembly occurs in the nucleolus.
• Telomeres and centromeres both contain heterochromatin.
• Telomeres shorten with each cell division and require telomerase to maintain their length.

Experimental Models

• Wild-type organisms have a fully functional, active gene and serve as a baseline or control.
• Knockout organisms have an intentionally inactivated or nonfunctional gene to identify its biological role.
• cDNA expression levels can vary based on cloning factors, but the resulting protein remains unchanged because the coding sequence is identical to the endogenous gene.