Lecture 17

Textbook Notes

17.2 RNA Processing in Eukaryotes

• primary transcripts — nonfunctional initial eukaryotic RNA

  • pre-mRNA — protein-coding genes

  • require multistep modifications, called RNA processing

• introns — regions of a gene that are transcribed but not represented in the final RNA

• exons — regions that are transcribed and represented in the final mature RNA

  • not just protein-coding regions

RNA splicing

• splicing — the process that removes the introns from the growing RNA strand

• small nuclear RNAs (snRNAs) catalyze splicing of primary transcripts working with a complex of proteins

• small nuclear ribonucleoproteins (snRNPs) — protein plus RNA macromolecular machines

• 4 steps of splicing

  1. snRNPs bind to the 5’exon-intron and 3’intron-exon boundaries, and also a region near the end of the intron with an adenine (A) ribonucelotide

  2. the spliceosome is assembled as more snRNPs join the complex

  3. the 5’ end of the intron is cut from the exon, and the intron forms a single-stranded stem plus a loop (a lariat) with the adenine at its connecting (branch) point

  4. the 3’ end of the intron is cut, releasing the intron as a lariat, and the exons are joined by a phosphodiester linkage. excised intron is degraded to ribonucleoside monophosphates

• many genes codes for RNA that can be spliced more than one way, allowing for the production of different proteins or mRNA from one gene

Adding Caps and Tails to Transcripts

• as soon as the 5’ end of a eukaryotic pre-mRNA emerges from RNA polymerase, enzymes add a 5’ cap

  • cap consists of a modified guanine nucleotide linked unusually, enabling ribosomes to bind to the mRNA, and protecting the 5’ end from being degraded by ribonucleases

• an enzyme cleaves the 3’ end of the pre-mRNA after a sequence called the poly(A) signal

Lecture Slides

• remember, the DNA strands are complementary, not identical, so different information is encoded in each strand

  • transcription: 3’ → 5’ on the template strand

  • RNA synthesis 5’ → 3’ adding nucleotides onto 3’ end

Termination of Transcription in E. coli

• Rho-dependent termination:

  • rho binds to RNA and transcription complex on the rho-recognition site

  • rho moves toward 3’ end

  • transcript dissociates from template strand; rho dissociates from RNAP

  • basically breaks the transcript from template strand and breaks of RNA polymerase in the process

• intrinsic termination (rho-independent)

  • coded into the DNA

  • AAAAA transcripts into UUUUU

  • creates a hairpin loop, stem and loop

  • coaxes the RNA and RNA polymerase off

RNA Types

• 3 classic RNA types, from 3 gene types

• key point: all genes are transcribed into RNA, but some RNAs are put to use immediately to make ANY protein, and some RNAs encode information used to make one specific type of protein

• messenger RNA (mRNA): these are the RNAs that are not functional by themselves, but instead carry the instructions for making specific proteins

  • nonfunctional information carrier, just a copy of the info

  • just gives the info to ribosomes

• ribosomal RNA (rRNA): these small RNA molecules form complexes with ribosomal proteins to make RIBOSOMES, which are the platforms on which protein synthesis occurs

• transfer RNA (tRNA): these small RNA molecules carry amino acids to the ribosomes, where only the amino acid gets incorporated into the growing protein chain

  • does not know which ribosome needs which amino acid, just kind of floats around

• all genes are transcribed by RNA polymerases

  • one RNA polymerase in bacteria

    • holoenzyme includes sigma subunit; core enzyme does not

  • three RNA polymerases in the nuclei of eukaryotes

    • RNA polymerase I: transcribes rRNA genes

    • RNA polymerase II: transcribes mRNA genes (and many others)

    • RNA polymerase III: transcribes tRNA genes, some rRNA

  • each recognizes its own type of promoter and no other, so they don’t get mixed up

• RNA pol I promoter: -90, -34 → rRNA

• RNA pol II promoter: -60, -30 → mRNA

• RNA pol III promoter: +30 (A-BOX), +60 (B-BOX) → tRNA

  • understands you need to start BEFORE promoter

Bacterial vs eukaryotic RNA polymerases

• sigma factor of bacterial RNA polymerase contacts DNA directly and binds to promoter sequence

• eukaryotic RNA polymerases have no sigma factors, and only weakly associate with DNA

• eukaryotes require accessory (regulatory) proteins called transcription factors (TFs)

  • TFs “invites” RNA polymerase to the DNA transcript

  • TFs begin formation of transcription initiation complex, which allows RNA polymerase to bind to promoter sequence

  • efficient transcription will NOT occur in absence of TFs in eukaryotes

Eukaryotic general transcription factors:

1. TFIID binds to TATA box in DNA (TFIID = type II TF for RNA pol II)

2. TFIIA and TFIIB form complex with TFIID

3. resulting complex is bound by RNA polymerase attached to TFIIF

4. preinitiation complex is completed by addition of TFIIE and TFIIH

5. RNA polymerase II undergoes phosphorylation (adds ATP, energy)

Genomic structure of bacteria and eukaryotes:

• most DNA in bacteria does code for proteins, rRNA or tRNA; coding sequences proceed without interruption

  • length of gene in DNA generally equal to length of RNA

• in eukaryotes, most DNA—between and within genes—does not code for proteins, rRNA or tRNA

  • called noncoding DNA

  • if this noncoding DNA is between genes: called spacer DNA

  • if this noncoding DNA is within a gene: called introns (“intervening” sequences)

  • coding regions within a gene: called exons (“expressed” sequences)

• for most eukaryotic genes, the length of the DNA sequence (the gene) is longer than the mRNA that participates in protein synthesis

• summary:

  • genes are recognized and transcribed into (primary) (initial) (pre-mRNA) transcripts

  • mRNA is processed into continuous coding sequence