1. Transcription 11/3/05

2. Stable RNA rRNA -Structural component of ribosomes
tRNA-Adaptors, carry aa to ribosome
Synthesis
Promoter and terminator
Post-transcriptional modification (RNA processing)
Evidence
Both have 5’ monophospates
Both much smaller than primary transcript
tRNA has unusual bases. EX pseudouridine

3. tRNA and rRNA Processing
Both are excised from large primary transcripts
1º transcript may contain several tRNA molecules, tRNA and rRNA
rRNAs simply excised from larger transcript
tRNAs modified extensively
5. Base modifications
Both are excised from large primary transcripts
1º transcript may contain several tRNA molecules, tRNA and rRNA
rRNAs simply excised from larger transcript
EX. Transcription of the rrnD operon yields a 30S precursor, which must be cut up to release the three rRNA and three tRNAs.
tRNAs modified extensively
EX. tRNATyr
Endonuclease cleaves ~200 bases from 3’ end
RNase D removes 7 bases from 3’ end
RNAse P generates 5’ end, leaving a monophosphate
RNase D generates 3’ CCA end
Extensive modification of bases, all in or near the loops

4. Examples of Covalent Modification of Nucleotides in tRNA
N6-Methyladenylate
(m6A)
N6-Isopentenyladenylate
(i6A)
Inosinate
(I)
7-Methylguanylate
(m7G)
tRNA modified bases
Dihydrouridylate
(D)
Pseudouridylate (Ψ)
(ribose at C-5)
Uridylate
5-oxyacetic acid
(cmo5U)
3-Methylcytidylate
(m3C)
Modifications are shown in blue.
5-Methylcytidylate
(m5C)
2’-O-Methylated nucleotide
(Nm)

5. Eukaryotic Transcription
Regulation very complex
Three different pols
Distinguished by -amanitin sensitivity
Pol I—rRNA, least sensitive
Pol II– mRNA, most sensitive
Pol III– tRNA and 5R RNA moderately sensitive
Each polymerase recognizes a distinct promoter
Regulation very complex
Three different pols distinguished by -amanitin sensitivity
Pol I—rRNA least sensitive
Pol II– mRNA most sensitive
Pol III– tRNA moderately sensitive
Each polymerase recognizes a distinct promoter
Promoters recognized
Pol I—bipartite promoter
Pol II– Upstream promoter
Pol III—internal promoter

6. Eukaryotic RNA Polymerases
Location
Products
-Amanitin Sensitivity
Promoter I
Nucleolus
Large rRNAs (28S, 18S, 5.8S)
Insensitive
bipartite promoter II
Nucleus
Pre-mRNA, some snRNAs
Highly sensitive
Upstream
III
tRNA, small rRNA (5S), snRNA
Intermediate sensitivity
Internal promoter and terminator
-amanitin used to distinguish different polymerases. Poison derived from mushrooms (Amanita sp.). How does this explain the reason mushrooms poison the way they do?

7. Eukaryotic Polymerase I Promoters
RNA Polymerase I
Transcribes rRNA
Sequence not well conserved
Two elements
Core element- surrounds the transcription start site (-45 to + 20)
Upstream control element- between -156 and -107 upstream
Spacing affects strength of transcription
RNA Polymerase I
Transcribes rRNA
Sequence not well conserved
Architecture well conserved
Two elements
Core element- surrounds the transcription start site
Upstream control element- ~ 100 bp upstream

8. Eukaryotic Polymerase II Promoters
Much more complicated
Two parts
Core promoter
Upstream element
TATA box at ~-30 bases
Initiator—on the transcription start site
Downstream element-further downstream
Many natural promoters lack recognizable versions of one or more of these sequences
Much more complicated
Two parts
Core promoter
Upstream element
TATA box at ~-30 bases
Initiator—on the transcription start site
Downstream element-further downstream
Many natural promoters lack recognizable versions of one or more of these sequences

9. TATA-less Promoters
Some genes transcribed by RNA pol II lack the TATA box
Two types:
Housekeeping genes ( expressed constitutively). EX Nucleotide synthesis genes
Developmentally regulated genes. EX Homeotic genes that control fruit fly development.
Specialized (luxury) genes that encode cell-type specific proteins usually have a TATA-box

10. mRNA Processing in Eukaryotes
Primary transcript much larger than finished product
Precursor and partially processed RNA called heterogeneous nuclear RNA (hnRNA)
Processing occurs in nucleus

11. Capping mRNA
5’ cap is a reversed guanosine residue so there is a 5’-5’ linkage between the cap and the first sugar in the mRNA.
Guanosine cap is methylated.
First and second nucleosides in mRNA may be methylated
1. 5’ cap is a reversed guanosine residue so there is a 5’-5’ linkage between the cap and the first sugar in the mRNA.
2. Guanosine cap is methylated.
3. First and second nucleosides in mRNA may be methylated
What is the function of the 5’ cap?
Clue: some viral mRNAs are not capped, yet they are translated efficiently and are stable. In these virus-infected cells the mechanism for initiation of mRNA translation is altered so that capped mRNAs are not translated efficiently!
BACK

12. Polyadenylation
Polyadenylation occurs on the 3’ end of virtually all eukaryotic mRNAs.
Occurs after capping
Catalyzed by polyadenylate polymerase
Polyadenylation associated with mRNA half-life
Histones not polyadenylated
Polyadenylation occurs uniquely on mRNA, not tRNA or ribosomal RNA. This allows simple and rapid purification of mRNA by hybridization to an affinity resin composed of oligo-dT. Histone mRNA is the only non-polyadenylated mRNA in the cell. Why is this??? May be related to histone synthesis uniquely during S-phase of the cell cycle.

13. Introns and Exons Introns--Untranslated intervening sequences in mRNA
Exons– Translated sequences
Process-RNA splicing
Heterogeneous nuclear RNA (hnRNA)-Transcript before splicing is complete
Introns--Untranslated intervening sequences in mRNA
Exons– Translated sequences
Process-RNA splicing

14. Splicing Overview Occurs in the nucleus
hnRNAs complexed with specific proteins, form a ribonucleoprotein particle (RNP)
Primary transcripts assembled into hnRNP
Splicing occurs on spliceosomes consist of
Small nuclear ribonucleoproteins (SnRNPs)
components of spliceosomes
Contain small nuclear RNA (snRNA)
Many types of snRNA with different functions in the splicing process
Occurs in the nucleus
hnRNAs complexed with specific proteins, form a ribonucleoprotein particle (RNP)
Primary transcripts assembled into hnRNP
Splicing occurs on spliceosomes consist of
Small nuclear ribonucleoproteins (SnRNPs)
components of spliceosomes
Contain small nuclear RNA (snRNA)
Many types of snRNA with different functions in the splicing process

15. Splice Site Recognition
Introns contain invariant 5’-GU and 3’-AG sequences at their borders (GU-AG Rule)
Internal intron sequences are highly variable even between closely related homologous genes.
Alternative splicing allows different proteins from a single original transcript
Precision is essential in splicing
Introns contain invariant 5’-GU and AG-3’sequences at their borders
Internal intron sequences are highly variable even between closely related homologous genes.

16. Simplified Splicing Mechanism
2’ hydroxyl group of an A nt within the intron attacks the phosphodiester bond linking the first exon to the intron. This attack breaks the bond between exon 1 and the intron, yielding the ree exon and a lariat exon-intron intermediate, with the GU linked to the internal A via a phosphodiester linkage to C-2 of ribose.
Free 3’ –OH on exon 1 attacks phosphodiester bond between intron and exon 2.
Yields spliced exon1-exon2 and lariat shaped intron. Phosphate at end of exon 2 becomes the phosphate linking the 2 exons.

17. RNA pol III Precursors to tRNAs,5SrRNA, other small RNAs
Promoter Type I
Lies completely within the transcribed region
5SrRNA promoter split into 3 parts
tRNA promoters split into two parts
Polymerase II-like promoters
EX. snRNA
Lack internal promoter
Resembles pol II promoter in both sequence and position
Precursors to tRNAs,5SrRNA, other small RNAs
Promoter Type I
Lies completely within the transcribed region
5SrRNA promoter split into 3 parts
tRNA promoters split into two parts
Polymerase II-like promoters
EX. snRNA
Lack internal promoter
Resembles polII promoter in both sequence and position

18. DNAse Footprinting Use: promoter ID End Label template strand
Add DNA binding protein
Digest with DNAse I
Remove protein
Separate on gel
Use: Promoter ID, identification of sequences involved in recognition by proteins
End Label template strand
Add DNA binding protein
Digest with DNAse I
Remove protein
Separate on gel
Run next to sequencing gel (Maxam-Gilbert Sequencing)
Will get all sizes represented unless they could not be made because the section was protected by a DNA binding protein.
Can focus on region of binding.
Protected region