RNA Processing and Capping

RNA Processing

• Almost all newly synthesized RNA molecules (primary transcripts) are processed to some degree in eukaryotic cells.
• Three major modifications occur:
  • The 5'-end is capped with methylguanosine.
  • Introns are spliced out.
  • Poly-A tail is built at the 3' end.

Capping the 5' of mRNA

• Capping protects mRNA from 5' exonuclease degradation.
• The cap is 7-methylguanosine linked to the 5' end of mRNA.
• Formed by condensation of GTP with the 5' end of mRNA.
• Guanine is then methylated.
• Occurs early in transcription.

Interrupted Genes in Eukaryotes: Exons & Introns

• Most eukaryotic genes contain non-coding sequences called introns that interrupt the coding sequences (exons).
• Introns are excised from the RNA transcripts prior to their transport to the cytoplasm.

Introns

• Introns are non-coding sequences located between coding sequences.
• Introns are removed from the pre-mRNA and are not present in the mRNA.
• Exons are composed of the sequences that remain in the mature mRNA after splicing.
• Introns are variable in size and may be very large.

Placing the Poly(A) Tail on the mRNA

• Pol II synthesizes RNA up to and beyond the highly conserved sequence: (5')AAUAAA.
• An endonuclease cleavage signal sequence is bound by an enzyme complex.
• The RNA is cleaved by the endonuclease at a point 10-30 nucleotides 3' to the sequence AAUAAA.
• The polyadenylate polymerase synthesizes a poly(A) tail.

Key Points

• Three different RNA polymerases are present in eukaryotes, and each polymerase transcribes a distinct set of genes.
• Eukaryotic gene transcripts usually undergo three major modifications:
  • Addition of 7-methylguanosine caps to 5' termini.
  • Excision of non-coding intron sequences.
  • Addition of poly(A) tails to 3' ends.
• All modifications occur in the nucleus prior to the transport to the cytoplasm.

Transcription in Prokaryotes: E. coli RNA Polymerase

Stages of Transcription

• RNA chain initiation.
• RNA chain elongation.
• RNA chain termination.

Promoters

• In E. coli, must be > 12 bp to avoid occurrence by chance.
• Promoters have only small conservation in sequence.
• Four main regions:
  • Startpoint.
  • -10 sequence (Pribnow box).
  • -35 sequences.
  • The 17 nucleotide spacer sequence between -10 and -35 sequences.

Numbering of a Transcription Unit

• The transcript initiation site is +1.
• Bases preceding the initiation site are given minus (-) prefixes and are referred to as upstream sequences.
• Bases following the initiation site are given plus (+) prefixes and are referred to as downstream sequences.

Starting Transcription

• Binding of RNA polymerase holoenzyme to a promoter region in DNA.
• Localized unwinding of both DNA strands (around -10 region) by RNA polymerase to provide a single-stranded template.
• Formation of phosphodiester bonds between the first few ribonucleotides in the nascent RNA chain.
• Conformational change in enzyme, promoter is cleared.
• Sigma factor is released.
• Nus A protein binds instead, ready for elongation "antitermination complex".

Termination of Transcription

• RNA polymerase transcribes until it meets a terminator.
• Transcription then stops and the RNA product disassociates from the DNA template.
• Many terminators are hairpin-forming sequences.
• Two types of terminators:
  • Rho-dependent terminators - require a protein factor (p).
  • Rho-independent terminators - do not require p.

E. coli RNA Polymerase

• Core enzyme: α β β'ω.
• Holoenzyme: α2 β β' ωσ.
• Functions of the subunits:
  • α: assembly of the tetrameric core.
  • β: ribonucleoside triphosphate binding site.
  • β': DNA template binding region.
  • σ: initiation of transcription.

Elongation

• RNA polymerase is bound to DNA and is covalently extending the RNA chain.
• Site for incoming ribonucleoside triphosphate.
• Growing RNA chain.
• Rewinding site.
• Short region of DNA/RNA helix.
• Unwinding site.
• Movement of RNA polymerase.

Transfer of Genetic Information: The Central Dogma

• Information stored in the nucleotide sequences of genes is translated into the amino acid sequences of proteins through unstable intermediaries called messenger RNAs.
• The mRNA codons on the mRNA are translated into an amino acid sequence by the ribosomes.

Transcription and Translation in Eukaryotes

• The primary transcript is pre-mRNA.
• A precursor to the mRNA.
• The pre-mRNA is modified at both ends, and introns are removed to produce the mRNA.
• After processing, the mRNA is exported to the cytoplasm for translation by ribosomes.

General Features of RNA Synthesis

• Similar to DNA synthesis except:
  • The precursors are ribonucleoside triphosphates.
  • Only 1 strand of DNA is used as a template.
  • RNA chains can be initiated de novo (no primer required).
• The RNA molecule will be complementary to the DNA template (antisense) strand and identical to the DNA non-template (sense) strand.
• RNA synthesis is catalyzed by RNA polymerases and proceeds in the 5'-3' direction.

The Central Dogma

Prokaryotes

• Gene
• DNA
• Transcription
• mRNA
• Translation
• Protein

Transcription vs. DNA Replication

• RNA does not remain H-bonded to the DNA post-synthesis.
• RNA molecules are selective copies of much shorter segments of the DNA.
• Both employ polymerases.
  • to make phosphodiester linkages.
  • DNA ahead of synthesis is unwound.
  • Building blocks are very similar to each other.
  • RNA polymerase does not need a primer.

The "Transcription Bubble"

• DNA 3'
• Rewinding
• Template strand
• Transcription bubble
• Nontemplate strand
• RNA
• RNA-DNA hybrid, -8bp
• Active site
• Direction of transcription
• Unwinding
• NTP channel