1. Posttranscriptional Modification

2. Eukaryotic mRNA modification
Prok mRNA is mature and ready for translation when it is synthesized
Euk mRNA requires modification so that it can be translated
Modification at the 5’ end
Modification at the 3’ end
Removal of introns and joining of exons

3. Eukaryotic mRNA modification
Euk mRNA requires modification so that it can be translated
mRNA cannot be exported from the nucleus until modified
Stabilizes/protects the mRNA from degradation; rapidly degraded when no 5’ or 3’ modification
Recognition of mRNA by ribosomes

4. Eukaryotic mRNA modification
5’ end modification—5’ capping
Addition of a ‘cap’ to the 5’ end of the mRNA
Added by capping enzyme
7-methyl guanosine (m7G)
Attached to the RNA via a 5’-5’ linkage
Nt at position 1 and 2 of the mRNA are methylated on the sugar group
Prevents degradation
Needed for recognition/binding by the ribosome

5. Fig. 11.8 Cap structure at the 5 end of a eukaryotic mRNA
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

6. Eukaryotic mRNA modification
3’ end modification—poly(A) tail
Addition of a string of adenines to the 3’ end of the mRNA; poly(A) tail
Usually adenines
No template is necessary to add the tail
Carried out in the nucleus by enzymes/protein complexes

7. Eukaryotic mRNA modification
3’ end modification—poly(A) tail
Recognizes the poly(A) consensus sequence (5’-AAUAAA-3’)
Found at the 3’ end in the 3’ UTR
Poly(A) addition site is usually nucleotides downstream of the poly (A) consensus sequence
Since no termination mechanism used during tc, the addition of the poly(A) tail is used to determine the length of the mRNA

8. Eukaryotic mRNA modification
3’ end modification—poly(A) tail
CPSF (cleavage and polyadenylation specificity factor) binds to the poly(A) consensus seq in the newly synthesized pre-mRNA
CstF (cleavage stimulation factor) binds to a GU- or U-rich region that is downstream to the poly(A) consensus seq in the pre-mRNA
CPSF and CstF bind to one another; loops the pre-mRNA

9. Eukaryotic mRNA modification
3’ end modification—poly(A) tail
CFI and CF II (CF=cleavage factor) bind pre-mRNA and cleave it at the cleavage site
PAP (poly(A) polymerase) binds and adds the adenines to the new end of the mRNA; ATP is the substrate
PABII protein (poly(A) binding protein II) is bound to the poly(A) tail

10. Fig. 11.9 Diagram of the 3 end formation of mRNA and the addition of the poly(A) tail
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

11. Eukaryotic mRNA modification
mRNA splicing
Euk RNA has introns (intervening sequences)
Noncoding
Interrupt the coding regions (exons)
Introns are removed
Exons joined back together
Carried out in the nucleus
Must be completed for mRNA export and translation

12. Fig. 11.10 General sequence of steps in the formation of eukaryotic mRNA
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

13. Eukaryotic mRNA modification
mRNA splicing
Carried out by spliceosome
complex of proteins and snRNAs
Called small nuclear ribonucleoprotein particles or snRNP’s (snurps)
5 main snRNA’s are U1, U2, U4, U5, U6
Introns are recognized by consensus sequences at the 5’ and 3’ splice junctions
5’-GUNNNNN
NNNNNNNNAG-3’

14. Eukaryotic mRNA modification
mRNA splicing
U1 snRNP binds 5’ splice junction
U1 base pairs with the splice junction—recognition
U2 snRNP binds branch point sequence where the 5’ end will bind to form the lariat
Branch point consensus seq—YNCURA
5’ binds to the A

15. Eukaryotic mRNA modification
mRNA splicing
U4/U6 binds to U5; U4/U6/U5 binds to U1 and U2 and RNA is looped to bring junctions close together
U4 dissociates
snRNPs cleave the intron at 5’ junction and it is bonded to the A in the branch point sequence—RNA lariat is formed
3’ junction is cleaved and the 2 exons are covalently joined together.

16. Fig. 5.12 Details of intron removal from a pre-mRNA molecule
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

17. Fig. 11.11 Model for intron removal by the spliceosome
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

18. Ribosome structure and rRNA
Ribosomes composed of 2 subunits
1 large subunit and 1 small subunit
Proks (E. coli): large subunit=50S and small subunit=30S; together they are 70S
Euks (mammals): large subunit=60S and small subunit=40S; together they are 80S
Subunits are composed of many proteins and at least 1 rRNA; rRNA is catalytic
Euk ribosomes are larger and more complex than prok ribosomes

19. Fig. 5.16 Composition of whole ribosomes and of ribosomal subunits in mammalian cells
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

20. Transcription of rRNAs--E. coli
rRNA genes are arranged together in the DNA to form transcription units
rRNA genes have some tRNA genes embedded in the transcription unit
Called rrn region; 7 of these regions in the E. coli genome

21. Transcription of rRNAs--E. coli
The rRNA is transcribed as one large piece—precursor rRNA (pre-rRNA)
Cleaved into mature rRNAs (16S, 23S, 5S, and tRNAs) by RNase III and other enzymes
Associate with ribosomal proteins as transcription is occurring.

22. Fig. 5.17 rRNA genes and rRNa production in E. coli
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

23. Transcription of rRNAs—Euks
rRNA genes are arranged together and repeated in tandem in the DNA times
Transcription at these regions produces the nucleolus (site of ribosomes assembly)
All but 5S rRNA are synthesized from this gene cluster; 5S is located elsewhere in the genome

24. Transcription of rRNAs—Euks
rRNA is transcribed by RNA polymerase I
Requires tc factors to bind to the DNA
Requires a promoter
Termination mediated by termination seq
The rRNA is transcribed as one large piece—precursor rRNA (pre-rRNA)
Cleaved into mature rRNAs (18S, 5.8S, 28S)
Associate with ribosomal proteins and assembled into ribosomes in nucleolus.

25. Fig. 5.18 rRNA genes and rRNA production in eukaryotes
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

26. Transcription of tRNAs—Euks
Carried out by RNA polymerase III
Tc tRNAs, 5S rRNA, and some snRNAs
Require tc factors to bind to DNA and promoter
tRNA genes are repeated in the euk genome
Each tRNA is unique but all have CCA added to 3’ end and are extensively modified posttranscriptionally

27. Transcription of tRNAs—Euks
tRNAs undergo extensive secondary structure—cloverleaf structure
Contains an anticodon that is complementary to the codon in mRNA.
Some tRNAs have introns that must be removed by splicing.

28. Fig. 5.21 Cloverleaf structure of yeast alanine tRNA
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

29. Fig Transcription factors involved in the initiation of human rDNA transcription by RNA polymerase I
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

30. Fig. 5.22a Three-dimensional structure of yeast phenylalanine tRNA as determined by X-ray diffraction of tRNA crystals
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

31. Fig. 5. 24 Cloverleaf models for yeast precursor tRNA
Fig Cloverleaf models for yeast precursor tRNA.Tyr and mature tRNA.Tyr
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

32. Fig. 5.1 Transcription process
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.