RNA Metabolism: Chapter 26

RNA Metabolism

• RNA plays three roles in protein synthesis: mRNA encodes amino acid sequence, tRNA reads mRNA and transfers amino acids, and rRNA is a constituent of ribosomes.

RNA Synthesis

• RNA synthesis is carried out by DNA-dependent RNA polymerase, which acts as a nucleophile attacking the phosphate of incoming rNTPs.
• Transcription resembles replication in its fundamental chemical mechanism, with polarity, template use, and initiation, elongation, and termination phases.
• Transcription differs from replication in not requiring a primer and involving only limited segments of a DNA molecule.

Initiation of Transcription

• Initiation starts at a promoter, where the DNA duplex unwinds, forming a transcription "bubble" of about 17 bp.
• The RNA-DNA hybrid occurs in this unwound region, and the transcription bubble moves along the DNA, generating waves of positive supercoils ahead and negative supercoils behind.

Elongation of Transcription

• Elongation of a transcript proceeds at a rate of 50-90 nucleotides/s.
• Movement of the transcription bubble requires considerable strand rotation of the nucleic acid molecules.
• Topological problems caused by transcription are relieved by topoisomerases.

RNA Polymerase

• RNA polymerase holoenzyme consists of five core subunits and a sigma factor, which directs the enzyme to specific binding sites on the DNA.
• The enzyme lacks 3'>5' proofreading activity.

Promoters

• E. coli promoters have several recognition sequences, including the -10 and -35 regions, and the UP element.
• The promoter region extends between positions -70 and +30.
• Variations in the consensus sequence affect the efficiency of RNA polymerase binding and transcription initiation.

Termination of Transcription

• Termination occurs in two ways: Rho-independent and Rho-dependent.
• Rho-independent termination occurs at characteristic sequences, including a stem and loop structure.
• Rho-dependent termination involves the Rho factor, which is an ATP-dependent helicase that associates with the RNA and migrates in the 5'>3' direction.

Eukaryotic RNA Polymerases

• There are three eukaryotic RNA polymerases: RNA pol I, RNA pol II, and RNA pol III.
• RNA pol I synthesizes pre-rRNA, which contains the precursor for the 18S, 5.8S, and 28S rRNAs.
• RNA pol II synthesizes mRNAs and requires an array of transcription factors.
• RNA pol III synthesizes tRNAs, the 5S rRNA, and some other small specialized RNAs.

Promoters in Eukaryotes

• Promoters in eukaryotes have a TATA box and an Inr region.
• The TATA box is a pyrimidine-rich sequence that is recognized by the TBP protein.
• The Inr region is a sequence that is recognized by the RNA polymerase.

RNA Polymerase II

• RNA pol II is a huge enzyme consisting of 12 subunits.
• The carboxyl-terminal domain (CTD) of the RPB1 subunit is phosphorylated by TFIIH during transcription initiation.

Transcription at RNA Polymerase II Promoters

• Transcription at RNA polymerase II promoters involves the sequential assembly of transcription factors, including TBP, TFIIB, TFIIF, and TFIIH.
• The DNA is unwound at the Inr region by the helicase activity of TFIIH, and the polymerase then escapes the promoter and begins elongation.

mRNA Modification in Eukaryotes

• mRNA modification in eukaryotes involves three steps: capping, splicing, and polyadenylation.
• Capping occurs before the synthesis of the primary transcript is complete.
• Splicing involves the removal of introns and the joining of exons to form a continuous sequence.
• Polyadenylation involves the addition of 80-250 A residues to the 3' end of the mRNA.

Modified RNA

• RNA polymerase can undergo selective inhibition by certain agents, including intercalating agents and rifampicin.
• Intercalating agents, such as actinomycin, intercalate into the double-helical DNA, deforming the DNA and inhibiting RNA elongation.
• Rifampicin inhibits bacterial RNA synthesis by binding to the B subunit of bacterial RNA polymerases, preventing the promoter clearance step of transcription.