Lecture 26: 10_25_06

mRNA Molecules in Prokaryotes are Polycistronic

• Many mRNA molecules in prokaryotes are polycistronic – that is, they encode two or more polypeptide chains.
• All mRNA molecules contain signals that define the beginning of each encoded polypeptide.
• The initiating codon in mRNA is AUG and the stop codon is UAA, UAG or UGA.
• The coding regions are referred to as Open Reading Frames (ORFs).
• A purine rich region at the 5’end of initiator codon is the Shine-Dalgarno sequence, which is the ribosome binding site.
• A Shine-Dalgarno sequence is present at the 5’end of each ORF, so the ORFs are translated separately.

RNA in Eukaryotes are spliced

• Genes in eukaryotes consisted of exons and introns.
• The exons are the coding regions while the introns are the non-coding regions.
• Introns must be excised and exons must be linked to form the final mRNA in a process called splicing.

• Each mRNA encodes a single polypeptide.
• Sequences in the introns mark the sites where splicing had to occur.
• Consensus motif is found in the 5’ and 3’ ends of introns referred to as the 5’ splice site and the 3’ splice site respectively.
• In addition, an internal site called the branch site is located between 20-50 nucleotides upstream of the 3’ splice site.

• A complex of small RNAs and proteins called spliceosome carries out the splicing of precursor mRNA.
• Splicing involves two steps:
1. Cleavage of phosphodiester bond between exon1 and the 5’end of intron. The attacking group is the 2’-OH group of an adenylate residue in the branch site, forming a 2’-5’ phosphodiester bond.
2. Joining of exon1 and exon2 as the free 3’-OH group of exon1 attacks the phosphodiester bond between the intron and exon2. The intron is released in lariat form.

§ Both steps are transesterification processes.

• The splicing of mRNA precursors is catalyzed by small nuclear RNAs (snRNAs).
• snRNAs are RNA molecules fewer than 300 nucleotides.
• Several snRNAs that are essential for splicing includes U1, U2, U4, U5 and U6, which associate with specific proteins to form complexes termed snRNPs (small nuclear ribonucleoprotein particles) pronounce as “snurps”.
• U1 snRNP binds the 5’ splice site and initiates the spliceosome assembly.
• U2 binds the branch site.
• Preassembled U4-U5-U6 tri-snRNP joins the U1, U2 and mRNA precursor to form the spliceosome.
• Rearrangement of RNA in the spliceosome facilitates the transesterification processes.

Catalytic RNAs

• RNAs are surprisingly versatile.
• RNAs can play catalytic role in biological processes as seen from the RNA molecules involved in splicing.
• Ribonuclease P is an enzyme that contains a key RNA component, which catalyzes the maturation of tRNA by endonucleolytic cleavage of nucleotides from the 5’ end of the precursor molecule.
• Another example is a ribosomal precursor from Tetrahymena (a ciliated protozoan) that splices itself in the absence of protein.
• Many of these catalytic RNAs also sometimes are referred to as ribozymes.