Week 6: Eukaryotic Transcription and Translation

Comprehensive practice flashcards covering eukaryotic transcription mechanisms, promoter architecture, RNA processing, and translation initiation, elongation, and termination based on lecture notes.

How do prokaryotes and eukaryotes differ regarding their default gene state, transcription/translation localization, and mRNA cistronic nature?

Prokaryotes have a default gene state of ON, carry out transcription and translation simultaneously in the same place, and produce polycistronic mRNA. Eukaryotes have a default gene state of OFF, carry out transcription and translation in different places at different times, and produce monocistronic mRNA.

Which types of RNA are transcribed by each of the three eukaryotic RNA polymerases?

RNA Polymerase I transcribes $47\text{S}$ pre-rRNA ($28\text{S}$, $5.8\text{S}$, and $18\text{S}$ rRNAs); RNA Polymerase II transcribes mRNAs, snRNAs, and scRNAs; RNA Polymerase III transcribes tRNAs, $5\text{S}$ rRNA, snRNAs, and scRNAs.

What are examples of constitutively silenced DNA and constitutively active DNA in eukaryotic cells?

Constitutively silenced DNA includes the inactive X-chromosome in female somatic cells (Barr Body), whereas constitutively active DNA includes housekeeping genes.

What role do pioneer transcription factors play in chromatin remodelling?

Pioneer transcription factors can bind to condensed DNA and facilitate cooperative binding to initiate local changes and chromatin relaxation at the gene site.

Which core promoter elements are recognized by basal transcription factors, and where are they typically located relative to the transcription start site ($+1$)?

The core promoter elements include BRE (B recognition element, located from $-37$ to $-32$), TATA box (located from $-31$ to $-26$), Inr (Initiator, located from $-2$ to $+4$), and DPE (Downstream promoter element, located from $+28$ to $+32$).

What are the common proximal promoter elements and their approximate upstream locations?

The proximal promoter elements include the GC box (located at approximately $-100$) and the CAAT box (located at approximately $-80$).

What is the composition and function of the TFIID complex?

TFIID is composed of TATA-binding protein (TBP) and $8\text{--}10$ TBP-associated factors (TAFs). It binds the TATA box, alters DNA conformation, associates with all three RNA polymerases, and serves as the functional equivalent of the bacterial sigma factor.

Which general transcription factors form the minimal transcription complex across the transcription start site?

TFIIA and TFIIB bind to the DNA minor groove to stabilise TFIID, and TFIIF binds downstream to recruit RNA Polymerase II, extending the complex across the transcription start site.

What roles does TFIIH play during transcription initiation?

TFIIH functions as a helicase that unwinds DNA ahead of the transcription complex, and it phosphorylates the C-terminal domain (CTD) tail of RNA Polymerase II (Ser-5 phosphorylation), which ejects mediator complexes and triggers productive elongation.

In which direction is the DNA template read, what is the rate of eukaryotic RNA synthesis, and which factors can pause elongation?

The antisense DNA strand is read $3'$ to $5'$ and RNA is synthesized $5'$ to $3'$ at a rate of $22\text{--}25\,\text{nt/sec}$. Elongation can be paused by DSIF (DRB-sensitivity inducing factor) and NELF (negative elongation factor).

What is the biochemical structure of the $5'$ mRNA cap?

A methylated guanosine triphosphate (GTP) is added $5'$ of base $1$ via a $5'\text{--}5'$ bridge to form the Cap0 structure; further methylation of nucleoside bases produces Cap1 or Cap2 structures.

Which sequence elements signal the termination of transcription for mRNA, and how is the transcript cleaved?

Key sequences include the polyadenylation signal (AAUAAA), polyadenylation site (CA), and a downstream GU-rich tract. Transcription proceeds beyond the gene, and endonucleases cleave the primary transcript before the GU-rich tract.

Which protein factors constitute the polyadenylation machinery, and how many adenine residues are added to the $3'$ end?

The complex includes CPSF (cleavage and polyadenylation specificity factor), CstF/CSF (cleavage stimulation factor), Poly(A) binding protein, and Poly(A) polymerase (PAP), which add $100\text{--}200$ adenine residues to the $3'$ end.

Which consensus sequences mark the boundaries of eukaryotic introns during pre-mRNA splicing?

The $5'$ splice donor site is marked by GU, the $3'$ splice acceptor site is marked by AG, and an internal branch site contains an invariant adenine residue ($\text{A}$).

What is the stepwise mechanism of intron removal carried out by the spliceosome?

Small nuclear RNAs (snRNAs) and snRNPs bind the transcript, the $5'$ splice site is cleaved, the released $5'$ end loops around to attach to the branch site forming a lariat structure, the $3'$ splice site is cleaved, and the adjacent exons are ligated together.

What trigger activates nonsense-mediated decay (NMD) in exported eukaryotic mRNA?

NMD is triggered when a premature stop codon is detected greater than $50\,\text{bp}$ ($>50\,\text{bp}$) upstream of the final exon-exon junction.

How are the four human rRNA genes organized, and by which RNA polymerases are they transcribed?

Humans have $>200$ rRNA genes. The $28\text{S}$, $5.8\text{S}$, and $18\text{S}$ rRNAs are clustered on acrocentric chromosomes and transcribed by RNA Polymerase I as a $47\text{S}$ precursor; the $5\text{S}$ rRNA is arranged in tandem arrays on chromosome 1q41-42 and transcribed by RNA Polymerase III.

What are the components of the eukaryotic 43S pre-initiation complex (PiC)?

The 43S PiC consists of the $40\text{S}$ small ribosomal subunit, ternary complex (eIF2 carrying GTP and Met-tRNA$_{\text{i}}^{\text{Met}}$), eIF1, eIF1A, and eIF3.

How do eukaryotic initiation factors loop the mRNA and recruit it into the ribosome?

eIF4 (with eIF4E binding the $5'$ cap and eIF4G binding poly(A)-binding protein, PABP) circularizes the mRNA into a loop, and eIF3 binds eIF4 to pull the looped mRNA into the ribosomal small subunit.

Which consensus sequence is scanned by eIF1 and eIF1A in the $5'\,\text{UTR}$ to identify the start codon?

They scan for the Kozak sequence, which has the consensus sequence GCCACCAUGG.

Which translation initiation factor mediates the joining of the 60S large ribosomal subunit to assemble the 80S ribosome?

eIF5 (specifically eIF5B-GTP) mediates the binding of the $60\text{S}$ large ribosomal subunit in an energy-dependent process accompanied by the release of initiation factors.

What are the three tRNA-binding sites on the large ribosomal subunit, and what occurs at each site?

The Aminoacyl (A) site presents charged incoming aminoacyl-tRNA to the mRNA codon; the Peptidyl (P) site holds the peptidyl-tRNA where peptidyl transferase links the peptide chain to the new amino acid; and the Exit (E) site is where uncharged tRNAs exit the ribosome.

Identify the four main structural arms and functional regions shown in this tRNA diagram:

The cloverleaf tRNA consists of the Acceptor arm ($3'$ end carrying the amino acid with a CCA sequence), the D loop/arm, the Anticodon loop/arm (which pairs with the mRNA codon), and the T (T$\Psi$C) loop/arm.

What post-transcriptional processing events are required to convert a primary eukaryotic pre-tRNA into a mature tRNA?

Cleavage of the $5'$ leader and $3'$ trailer sequences by endo- and exo-ribonucleases, addition of CCA to the $3'$ end by tRNA nucleotidyl transferase, splicing of internal introns, and chemical base modifications (such as pseudouridine and dihydrouridine).

What causes translation termination when a ribosome encounters a stop codon (UAA, UAG, or UGA)?

Stop codons do not encode an amino acid and have no complementary tRNA; instead, protein release factors bind to the ribosomal A site, hydrolyze the ester bond linking the polypeptide to the tRNA in the P site, and trigger ribosomal dissociation.

What is codon bias, and how do choke points and slippery sequences affect translation rate?

Codon bias is the organism-specific preference for certain synonymous codons based on tRNA availability. Uncommon codons create choke points that slow translation due to scarce tRNAs, while abundant/preferred codons create slippery sequences that accelerate translation.

What are notable differences between the standard nuclear genetic code and the mammalian mitochondrial (mtDNA) code?

In mammalian mtDNA, UGA encodes Tryptophan instead of a STOP codon; AGA and AGG code for STOP instead of Arginine; and AUA/AUU code for Methionine instead of Isoleucine.

How are proteins destined for secretion or the endomembrane system targeted to the rough endoplasmic reticulum?

They contain an N-terminal signal sequence that is recognized by Signal Recognition Particles (SRP), which bind the translating ribosome and dock it onto the rough ER membrane.