Transcription

1
Transcription
2
Transcription

• Transcription- the synthesis of RNA using DNA as
  a template.
• Four stages Initiation, Elongation,
  Termination, Post-transcriptional modification

3
Similarities and Differences from Replication

• Similarities
• 5' ? 3' direction
• many proteins involved
• initiation, elongation, termination
• transcription bubble
• starts and stops at specific places
• Nt not dNt

• Differences
• RNA polymerase instead of DNA polymerase
• no proofreading
• posttranscriptional modification
• 1 strand copied not 2
• not all copied

4
Terminology of Transcriptionsee Fig. 26-5
Lehninger POB 4th Ed.
5
Initiation
6
Transcription Start Site is Promotersee Fig.
26-4 Lehninger POB 4th Ed. (UP)
7
RNA Polymerase DNA-Dependent RNA PolymeraseFig.
26-4 Lehninger POB 3rd Ed.

• zinc metalloenzyme
• ??ßß? core enzyme (390 kDa)
• with ? subunit, holoenzyme (?70 most common)
• other subunits
• the ? subunit causes specific binding to promoter
• also dependent on NTPs (and Mg2 as usual)
• no requirement for primer
• No proofreading mistake every 104-105 bases

8
Elongation
9
Termination

• Rho (r)-dependent vs. r-independent

10
r-Independent Termination

• 5-(N)n CCCAGCCCGCCUAAUGAGCGGGCUUU

11
r-Dependent Termination

• A hairpin forms
• if protein called r is present, polymerase
  detaches
• Dissociation is somehow coupled to ATP hydrolysis

12
Comparison with Eukaryotes

• Prokaryotes Eukaryotes
• promoter promoter enhancers
• Polymerase Polymerases I, II and III
• Rho not Rho
• not as much processing Processing
• 50-90 nt/sec Slower

13
Eukaryote Upstream
14
Animal RNA Polymerases
15
Primary Transcript

• Primary Transcript- the initial molecule of RNA
  produced. AKA hnRNA
• hnRNA-heterogenous nuclear RNA
• In prokaryotes, DNA ? RNA ? protein in cytoplasm
  concurrently
• In eukaryotes nuclear RNA gtgt Cp RNA

16
Post-transcriptional Processing of mRNA Occurs in
Nucleus

• Cap
• functions transport, recognition, prevents
  exonucleases
• poly A tail
• functions prevents exonucleases, recognition,
  other?
• Less in prokaryotes
• Introns spliced out- eukaryotes only

17
Post-transcriptional Processing of mRNA -
Cappingsee Fig. 26-12 Lehninger POB 4th Ed.
18
Post-transcriptional Processing of mRNA -
Methylating the Capsee Fig. 26-12 Lehninger POB
4th Ed.
19
Methylation of the 2-OH of First (and Second)
Base Can Also Occursee Fig. 26-12 Lehninger POB
4th Ed.
20
Post-transcriptional Processing of mRNA -
Polyadenylationsee Fig. 26-18 Lehninger POB 4th
Ed.

• Prokaryotes n20-50
• Eukaryotes n80-250
• of prokaryotic mRNAs have polyA tail
• 100 of eukaryotic mRNAs have polyA tail

21
Post-transcriptional Processing of mRNA -
Splicing out Introns

• Complicated
• snurps - small nuclear ribonucleoproteins
• snurps 1-6 bind at specific times
• RNA catalysts
• lariat structure

22
Intron Groups and Characteristics
23
Group II Intron Consensus Sequence
intron
24
Group II Intron Splicing

• U1 base-pairs first by 5 end of intron

25
Group II Intron Splicing

• U1 base-pairs first by 5 end of intron

26
Group II Intron Splicing

• U1 base-pairs first by 5 end of intron
• U2 binds branch site and directs U1 binding there

27
Group II Intron Splicing

• U1 base-pairs first by 5 end of intron
• U2 binds branch site and directs U1 binding there
• U5 and U4/U6 then bind
• U4 released
• U2/U6 causes cleavage?

28
Group II Intron Splicing

• U1 base-pairs first by 5 end of intron
• U2 binds branch site and directs U1 binding there
• U5 and U4/U6 then bind
• U4 released
• U2/U6 causes cleavage?

29
Typical Structure

• Typical exon size lt 1000 nt
• Many 100-200 nt
• So 30-60 amino acids long
• typical intron highly variable
• 50-20,000 nt

30
Processing of rRNAsee Fig. 26-21 Lehninger POB
4th Ed.

• RNA synthesized longer than needed (polytenes)
• structural portions methylated
• non-methylated parts degraded
• RNA associates with protein
• final shape globular

31
Processing of tRNA

• Synthesized longer than needed

32
Processing of tRNAFig. 26-23 Lehninger POB 4th
Ed.

• Synthesized longer than neededtrimmed

33
Processing of tRNAFig. 26-23 Lehninger POB 4th
Ed.

• Synthesized longer than neededtrimmedspliced

34
Processing of tRNAFig. 26-23 Lehninger POB 4th
Ed.

• Synthesized longer than neededtrimmedsplicedCCA
  added

35
Processing of tRNAFig. 26-23 Lehninger POB 4th
Ed.

• Synthesized longer than neededtrimmedsplicedCCA
  addedand bases modified.

36
Base ModificationFig. 26-24 Lehninger POB 4th Ed.
37
Final Structure Cloverleaf Fig. 27-13
Lehninger POB 4th Ed.
38
Advanced Topics

• Regulation of mRNA half-life
• Alternative splicing

39
mRNA Half-life

• t½ seconds if seldom needed
• t½ several cell generations (i.e. 48-72 h) for
  houskeeping gene
• avg 3 h in eukaryotes
• avg 1.5 min in bacteria
• PEPCK
• Insulin 30 min
• -Insulin 3 h

40
mRNA degrades by RNase

• Exonuclease
• Major p/w
• Uses 53
• Deadenylate
• Decap
• Degrade
• 3 sequence inhibits (PEPCK)

41
Alternative SplicingFig. 26-20 Lehninger POB 4th
Ed.
42
VDJC Sequence Light ChainFig. 5-1 Stites, Stobo,
and Wells Basic and Clinical Immunology 6th Ed.
43
Eukaryote Upstream

• Promoter tells where to start
• Boxes tell how often to start
• TATA box has many proteins associated TBP (TATA
  binding protein), TFII_, TAFs (TBP associated
  factors)

44
Boxes and FactorsHarpers Review of Biochemistry

• Cis- vs. Trans-acting factors

45
Enhancers Work Upstream, Downstream or in the
Middle of a Gene

• They also work forwards or backwards
• Possible ways of working
• Different transcription factors
• Order of binding (differing concentrations)
• Affinity of transcription factors