RNA Processing II: Capping & Polyadenylation

This set of practice flashcards covers the molecular biology of RNA processing, specifically the mechanisms, enzymatic steps, functions, and signaling motifs involved in mRNA capping and polyadenylation.

When exactly does cap synthesis occur during the transcription process?

Cap synthesis occurs early in transcription before the chain is $30\,nt$ long.

What is the specific chemical structure of the RNA cap at the $5'\text{-terminus}$?

The cap is made of $7\text{-methylguanosine}$ ($m^7G$) linked via a triphosphate linkage.

What are the four primary functions of mRNA caps?

1. Protect mRNAs from degradation. 2. Enhance translatability. 3. Transport of mRNAs out of the nucleus. 4. Efficiency of splicing mRNAs.

According to the 1974 findings, where is methylation specifically clustered in eukaryotic mRNA?

Methylation is clustered at the $5'\text{-end}$ of the mRNA, forming the cap structure.

What are the four enzymatic steps of cap synthesis?

a) RNA triphosphatase removes terminal phosphate from pre-mRNA; b) Guanylyl transferase adds capping GMP from GTP; c) Methyl transferase methylates $N^7$ of capping guanosine; d) Methyl transferase catalyzes $2'\text{-O-methylation}$ of the penultimate nucleotide.

Which enzyme adds the poly(A) tail post-transcriptionally, and which molecule is it not encoded in?

The poly(A) tail is added by poly(A) polymerase and is not encoded in the DNA.

What is the typical length of the chain of AMP residues at the $3'\text{-ends}$ of eukaryotic mRNAs?

Approximately $250\text{ nucleotides}$ long.

What three motifs comprise a minimum efficient mammalian polyadenylation signal?

An $\text{AAUAAA}$ motif about $20\,nt$ upstream, followed $23$ or $24\,bp$ later by a $\text{GU-rich}$ motif, followed immediately by a $\text{U-rich}$ motif.

Which six proteins are required for the cleavage of pre-mRNA in mammals?

1. CPSF (Cleavage and Polyadenylation Specificity Factor); 2. CstF (Cleavage Stimulation Factor); 3. CF I (Cleavage Factor I); 4. CF II (Cleavage Factor II); 5. Poly(A) polymerase; 6. RNA polymerase II.

What is the specific role of Poly(A)-Binding protein II (PAB II) in polyadenylation?

PAB II binds to a pre-initiated oligo(A) and aids poly(A) polymerase in elongating the tail to $250\,nt$ or more, acting independently of the $\text{AAUAAA}$ motif.

At what point does polyadenylation become independent of the $\text{AAUAAA}$ signal during initiation?

When the poly(A) reaches about $10\,nt$ in length.

What is the function of the carboxy-terminal domain (CTD) of the $Rpb1$ subunit of RNA polymerase II?

The CTD serves as a platform where the capping, polyadenylating, and splicing enzymes bind directly to.

What are the two steps involved in the mechanism of transcription termination by RNA polymerase II?

1. Co-transcriptional cleavage (CoTC) within the termination region downstream of the polyadenylation site; 2. Cleavage and polyadenylation at the $\text{poly(A)}$ site, signaling the polymerase to dissociate.

In the Reovirus cap structure, what are the specific charges contributed by the triphosphate linkage and the terminal phosphate?

The triphosphate linkage contributes three negative charges, and the terminal phosphate contributes two negative charges.