Transcription/RNA processing basics

Central Dogma – information flows one way, DNA -> RNA -> protein

• Transcription/translation

RNA polymerase

-          making an RNA complementary to template DNA

-          enzyme RNA polymerase (RNAP) reads template sequence, synthesizes complementary strand

o       RNAP of plant, animal, bacterial, archeae all somewhat similar

§         Two large subunits which catalyze addition of ribonucleoside triphosphate

§         Additional smaller subunits

·         Bacteria have 5 subunits

·         Eukaryotes have 12 to 15 or so

-          RNA polymerization (Fig. 6.17 shows DNA polymerization)

o       nucleophilic attack of 3’ O(H) on the a–phosphate of a ribonucleoside triphosphate

o       so RNA is made 5’ to 3’, RNAP reads 3’ to 5’

o       formation of new phosphodiester bond and release of pyrophosphate

§         this furnishes energy, in principle no other energy source required (ie DG is negative) (just like DNA polymerase)

Transcription

-          stages in transcription (Fig. 8.11)

o       initiation

§         RNAP binds at DNA sequence called promoter (closed complex)

·         Bacterial

o       Sigma subunit binds to -10 and -35 boxes (upstream) (Fig. 8.12)

·         Eukaryotic

o       No sigma subunit, is recruited to promoter by other proteins

§         open complex formation (duplex melts around start site)

§         formation of first few phosphodiester bonds

·         in bacteria, sigma subunit released now

o       elongation

§         transcription bubble moves along with RNAP (downstream)

§         about 14bp

§         about 8 RNA/DNA bp

§         once one RNAP clears promoter region, another can bind and start

o       termination

§         release of primary transcript

§         usually happens at specific sequences

·         often a hairpin formed in RNA that causes release

RNA processing

eukaryotic primary transcripts (pre-mRNAs) are processed in three ways before they leave the nucleus

1) message is capped at 5’ end immediately when nascent end emerges

o       several enzymes put on 7-me G via a 5’-5’ linkage

3) message is polyadenylated at 3’ end (last RNA processing event)

o       enzyme cuts after a particular sequence (AAUAAA) (so new 3’ end)

o       poly(A) polymerase adds 100-250 A’s in a row (nontemplated)

2) message is spliced

o       in late ‘70s found that if you hybridize viral mRNA to DNA, get big loops in DNA

§         suggests that parts of RNA cut out after transcription

o       DMD gene

§         Transcribed gene is 2.5 million bp long

§         Primary transcript is 2.5 million nt

§         mRNA (after processing) is 14,000nt

§         so 99.5% of primary transcript is cut out

§         this is extreme example, but it is typical that 95% of primary transcript is cut out

o       sequences found in mRNA are exons (exported from nucleus)

o       sequences cut out are intron

o       typical eukaryotic genes have many small (a few hundred nt) exons and larger (thousands of nt) introns

§         DMD mean exon is 200nt, mean intron is 35knt)

o       Rules governing how many introns, where they are, and what purpose is are a mystery

Mechanism of splicing

• Important sequences
  • Three important sequences (Fig. 8.16 shows)
    • 5’ splice site
      • GU first two nt of intron
    • Branch point
      • Always has an A in it
    • 3’ splice site
      • intron ends in AG
      • pyrimidine-rich region just upstream
      • usually around 20-50nt downstream of branch point
• The reaction
  • First the transcript is cut (phosphodiester bond broken) at 5’ splice site
  • 5’ end of intron curls around and makes 2’-5’ phosphodiester bond with branch site A
  • Second cut (phosphodiester bond broken) at the 3’ splice site
  • Two exons are joined together
  • Intron is excised as a lariat structure
• The enzymes
  • A large complex called the spliceosome carries out splicing
  • Spliceosome
    • snRNPs – small nuclear ribonucleoproteins + 50 other proteins
      • these are a short RNA interacting with proteins to form a part of an enzyme
      • RNA in snRNP basepairs with primary transcript, that’s how spliceosome finds splice sites and branch point

Alternative splicing

Antibody produced by B cells

• Antibody is a protein
• Sometimes is a transmembrane protein, sometimes is secreted into blood
• Alternative splicing of “heavy chain” gene determines which form is made by cell
• Fig. 8.18
  • In exon 6 there is a splice site that is only used sometimes
  • If splicing does NOT happen here (right side of figure), exons 7 and 8 do not get on mRNA
    • Makes form of protein that is secreted
  • If 5’ splice site in middle of exon 6 is used, then exons 7 and 8 are added to mRNa
    • Makes form of protein that is attached to membrane, because the part of the protein encoded by exons 7/8 is a transmembrane domain.

Questions

Try questions 18-20 in the back of the chapter.  I’m not thrilled with these, but they’ll do for now.  They completely ignore most of the stuff on RNA processing, which is half of what I covered.