Gene Expression, Replication and DNA Repair

RNA interference

RNA interference (RNAi) is a sequence-specific gene-silencing pathway in which double-stranded RNA is processed into short guide RNAs that direct the RNA-induced silencing complex (RISC) to complementary transcripts. The effector complex is RISC, and it blocks gene expression by cleaving the target mRNA or repressing its translation. Editing, polyadenylation and splicing modify or process RNA, but they do not use RISC to silence genes.

The term is often confused with related small-RNA pathways.

• siRNA — a short double-stranded RNA, usually of exogenous or viral origin, that is loaded into RISC.
• miRNA — an endogenous small RNA encoded in the genome. In animal cells it pairs imperfectly with the 3' untranslated region of target mRNAs and represses translation or promotes decay.
• piRNA — a small RNA of roughly 24 to 31 nucleotides that binds PIWI-clade proteins in germ cells and silences transposable elements. The name refers to the PIWI partner, not to a P-element.

Biogenesis of miRNA requires two RNase III enzymes. Drosha cleaves the primary transcript in the nucleus, and Dicer cleaves the hairpin precursor in the cytoplasm. Cas9, a bacterial nuclease used in genome editing, and XRCC4, a double-strand break repair factor, are not involved.

Example: a miRNA that pairs with the 3' UTR of a target mRNA reduces its protein output without changing transcription of the gene.

mRNA reading frame and codons

A codon is a triplet of nucleotides in mRNA that specifies one amino acid or a stop signal. Translation starts at an AUG start codon and proceeds in triplets until one of the stop codons UAA, UAG or UGA is reached. Stop codons are part of the reading frame, but they do not specify an amino acid. Therefore a reading frame that contains n sense codons and one stop codon has n+1 codons but only n amino acids.

A single mRNA can contain several potential reading frames. To find the longest possible polypeptide, each start codon should be followed through its frame to the first in-frame stop.

• Open reading frame — the stretch from a start codon to the first in-frame stop codon.
• Non-standard code — some mitochondrial codes read additional codons as stops, which shortens the polypeptide made in that compartment.

Classifying point mutations depends on the codon change. A substitution that changes a sense codon to a stop codon is a nonsense mutation, and a change that alters an amino acid is missense. A purine-to-pyrimidine or pyrimidine-to-purine change is a transversion, and a purine-to-purine or pyrimidine-to-pyrimidine change is a transition.

Example: in a UAC (Tyr) codon, a C to A change gives UAA, which is a stop. This is a nonsense mutation and a transversion.

Eukaryotic RNA polymerases

Eukaryotic nuclei contain three main RNA polymerases, each specialised for a class of genes. RNA polymerase I transcribes the rRNA genes that give the large precursor of 28S, 18S and 5.8S rRNA in the nucleolus. RNA polymerase II transcribes protein-coding genes and most small nuclear RNAs. RNA polymerase III transcribes tRNA genes and the 5S rRNA gene.

All of these enzymes are DNA-dependent RNA polymerases, meaning they copy a DNA template into RNA. This is the basic chemistry of transcription, and it differs from the other enzyme classes below.

• DNA-dependent DNA polymerase — copies DNA into DNA, as in replication.
• RNA-dependent DNA polymerase — reverse transcriptase, which copies RNA into DNA.
• RNA-dependent RNA polymerase — copies RNA into RNA, as in RNA viruses.

Eukaryotic transcription of nuclear genes is therefore performed by DNA-dependent RNA polymerases. Plant-specific polymerases IV and V have roles in silencing and are not the enzymes that transcribe rRNA precursors.

Example: a mRNA encoding a cytosolic enzyme is transcribed by RNA polymerase II, whereas tRNA is transcribed by RNA polymerase III.

Lac operon regulation

The lac operon of E. coli is controlled by two independent signals. The LacI repressor binds the operator and blocks RNA polymerase. When lactose is present, its isomer allolactose binds the repressor, releases it from the operator and allows transcription. Lactose alone is therefore an inducer of the repressor switch, but it does not by itself give full expression.

The second signal is catabolite repression. Glucose keeps intracellular cAMP low, so the cAMP-CAP complex is not formed and the promoter is weakly activated. When glucose is absent, cAMP rises, cAMP-CAP binds the promoter and promotes RNA polymerase binding. Full induction therefore requires lactose present and glucose absent.

• Repressor-operator — absence of an inducer keeps the operon off.
• CAP-cAMP — a positive activator that is needed for strong transcription when glucose is low.

Example: an E. coli culture with both glucose and lactose uses glucose first, because high glucose keeps the operon weakly expressed even when lactose is present.

Replication and polymerase enzymes

Different polymerases differ in the type of template they read and the type of product they make. The four types are DNA-dependent DNA synthesis, DNA-dependent RNA synthesis, RNA-dependent DNA synthesis, and RNA-dependent RNA synthesis.

• Primase — synthesises short RNA primers by copying a DNA template, so it is DNA-dependent RNA synthesis.
• Reverse transcriptase — copies an RNA template into DNA, so it is RNA-dependent DNA synthesis.
• RNA replicase — copies an RNA template into RNA, so it is RNA-dependent RNA synthesis.
• DNA polymerase III — the main replicative polymerase of E. coli, which copies DNA into DNA.

In bacterial replication, the RNA primer made by primase is removed by DNA polymerase I. Its 5' to 3' exonuclease activity excises the primer, and its polymerase activity fills the gap with DNA. DNA polymerase III lacks this excision activity, and DNA gyrase acts ahead of the fork to relieve supercoiling.

Example: primase makes an RNA primer with a free 3'-OH, DNA polymerase III extends from it, and DNA polymerase I later replaces the primer with DNA.

DNA mismatch and base excision repair

Spontaneous deamination converts adenine to hypoxanthine. Hypoxanthine pairs with cytosine instead of thymine, so the lesion creates a mismatch that must be corrected. The correction is performed by base excision repair (BEx), which removes the altered base and replaces it with the correct nucleotide.

The pathway uses four activities. A DNA glycosylase cleaves the N-glycosidic bond to remove the damaged base, an AP endonuclease (a nuclease) cleaves the sugar-phosphate backbone at the abasic site, DNA polymerase fills the gap with the correct nucleotide, and DNA ligase seals the remaining nick.

Replication enzymes are not repair enzymes. Primase makes primers and helicase unwinds the duplex, and neither removes a mispaired base. Keep these apart when a question asks for the enzymes of a repair pathway.

Example: deamination of adenine in a duplex is corrected by glycosylase excision followed by nuclease incision, polymerase synthesis and ligation.

Alternative splicing

Alternative splicing is the selection of different combinations of exons from a single pre-mRNA. It allows one gene to produce several protein isoforms. Differences in gene expression between cell lineages, such as neurons and hepatocytes, arise from lineage-specific transcription factors and regulatory networks, so the assertion that cells of different lineages express different gene sets is true.

However, alternative splicing is not the only source of protein diversity. Other mechanisms include RNA editing, the use of alternative promoters or transcription start sites, and post-translational modification of proteins. A statement that calls alternative splicing the only mechanism is therefore false.