1. Transcription and the Central Dogma
1961: DNA is the molecule which stores genetic information, but
DNA is in nucleus, ribosomes (where protein synthesis takes place) are in the cytoplasm.
RNA is synthesized in the nucleus, is similar to DNA.
RNA migrates to cytoplasm (where ribosomes are)
Amount of RNA generally proportional to amount of proteins in the cell.
All this suggests role of RNA as messenger.

2. About RNA 1) DNA is double stranded, but RNA is single stranded.
However, RNA can base-pair with itself to create double stranded regions.
RNA
DNA
tRNA
genetics.gsk.com/graphics/ dna-big.gif

3. About RNA-2 2) RNA contains ribose instead of deoxyribose
3) RNA contains uracil instead of thymine.
deoxyribose.htm

4. 3 kinds of RNA
mRNA: a copy of the gene; is translated to make protein.
tRNA: smallest RNA, does actual decoding.
rRNA: 3 sizes that, along with proteins, make up a ribosome.
tRNA
rRNA
Tobin and Duschek, Asking About Life;

5. More kinds of RNA snRNAs snoRNAs Other RNAs, e.g. Xist
Small RNAs ( bases) that attach to proteins to form snRNPs (small nuclear ribonucleoproteins)
snRNPs are components of the spliceosome
Removes introns from pre-mRNA
snoRNAs
Small nucleolar RNAs, process rRNA
Modify other RNAs; guide RNA of telomerase
Other RNAs, e.g. Xist

6. All RNA comes from the DNA
mRNA, obviously
tRNA, rRNA
Telomerase guide RNA
Xist
antisense-RNA
Thus, we talk about RNA genes, sections of DNA that code for RNAs

7. Transcription: making mRNA
RNA a polymer assembled from monomers
Ribonucleoside triphosphates: ATP, UTP, GTP,CTP
RNA polymerase
Multi-component enzyme
Needs a template, but NOT a primer
In bacteria, a component (sigma) recognizes the promoter as the place on DNA to start synthesis
Synthesis proceeds 5’ to 3’, just as in DNA
mRNA is complementary and antiparallel to the DNA strand being copied.

8. Transcription-2
The order of nucleotides in the RNA reflects the order in the DNA
If RNA is complementary to one DNA strand, then it is identical (except for T change to U) to the other DNA strand.
Either DNA strand may contain the gene! Transcription just runs the other direction.

9. Sense, antisense Compare the sense strand of the DNA to the mRNA.
Note that mRNA synthesis will be 5’ to 3’ and antiparallel.

10. More about RNA polymerase
In bacteria, components are ββ’α2φσ
RNA polymerase is processive; once enzyme attaches to DNA, it can copy >10,000 nucleotides without falling off.
In eukaryotes, there are 3 RNA polymerases:
One for rRNA
One for tRNAs and some rRNA
One for all mRNAs and some small RNAs (involved in RNA processing)

11. Transcription needs a Promoter
A promoter is non-transcribed DNA
Prokaryotes
Eukaryotes

12. The Process of Transcription
Promoter recognition: 2 consensus sequences
The -10 region: TATAAT (10 bases upstream from where transcription actually starts.
The -35 region, farther upstream, also important.
“Consensus” sequence meaning the DNA sequence from many genes averages out to this.
The closer these 2 regions actually are to the consensus sequences, the “stronger” the promoter, meaning the more likely RNA polymerase binding and transcription will occur.

13. Consensus sequence
Numbers indicate the percentage of different genes in which that nucleotide appears in that spot in the promoter sequence.

14. The Process of Transcription-2
After binding to the promoter, polymerase “melts” DNA, lines up first base at the +1 site = Initiation.
RNA synthesis continues (Elongation), only the template strand being transcribed.
Termination: must be a stop sign, right?
In bacteria, hairpin loop followed by run of U’s in the RNA. Of course, the DNA must code for complementary bases and a run of A’s. See next.
Termination factor “rho”. Accessory protein.

15. Termination of Transcription in Bacteria
The hairpin loop destabilizes the interactions between the DNA, mRNA, and polymerase; U-A basepairs are very weak, and the complex falls apart.
In euks, termination occurs with a processing step.

16. About mRNA structure, etc.
Start site of transcription is NOT equal to start site of Translation
First codon read, AUG, is downstream from the first ribonucleotides. +1 is transcription start, not translation start.
AUG marks the beginning of an Open Reading Frame (ORF).
Lifetime of a eukaryotic mRNA is variable
For prokaryotes, mRNA is short lived, fits in with need of microbes to respond quickly to changes in environment.

17. Eukaryotic transcription
Occurs in nucleus, then mRNA goes to cytoplasm
Promoters but also enhancers
Enhancers also segments of DNA
Eukaryotic RNA requires processing
Pre-mRNAs found in nucleus as hnRNA
Heterogeneous nuclear RNA
hnRNPs: heterogeneous nuclear ribonucleoproteins

18. Initiation of transcription (euks)
Goldberg-Hogness (TATA) box at -35
CAAT box at -80 (non specific, but important)
GGCCAATCT consensus sequence
Enhancers
Elements of DNA that promote transcription
Can be upstream, downstream, even in gene

19. Eukaryotic RNA polymerase
A 10 subunit machine
Makes several attempts, abortive transcription
Key: successful synthesis of short DNA-RNA hybrid, stabilizes association of Pol with DNA
Then, processive to a high degree, transcribes until knocked off by termination signal.

20. Processing of mRNA Cap 7-methyl guanosine is added as a cap, 5’ to 5’
Cap aids in binding of mRNA to the ribosome
Shine-Delgarno seq. does same in prokaryotes

21. Processing of mRNA-2 Poly-A tail
In several steps, end of mRNA is cleaved off, and several rounds of AAAAAAA are added
Poly-A tail improves stability of mRNA, resists degradation by nucleases in cell.
departments.oxy.edu/.../ processing_of_hnrnas.htm

22. Introns and Exons
Introns are intervening sequences that do not contain information for making the protein.
Exons are the coding sequences left behind.

23. Removal of Introns Three types of intron-removal mechanisms
Spliceosomes:
hnRNA or pre-mRNA (to be processed)
snRNPs: small nuclear ribonucleoproteins
snRNAs: involved structurally and catalytically
In some systems, intron codes for protein that splices out the intron!
In Tetrahymena, intron is self-splicing: ribozyme

24. Splicing of Introns
In most systems, snRNPs cut mRNA, introns come out, and snRNPs help splice exons back together.
Particular sequences in the mRNA mark the beginning, end of exons and introns so snRNPs can do their job.

25. Introns:
How were they discovered? RNA-DNA hybrids weren’t colinear- loops of DNA extend out where there is no RNA to base pair with it. RNA = red.
Mutations in introns: don’t have much affect unless:
Mutation is near a splice site
Mutation is in a regulatory region (which could be in an intron)
There is a separate gene within the intron.

26. Why are there introns? Very ancient?
Located in the same positions in genes common to plants and animals. Maybe bacteria had them once and lost them.
Self-splicing RNAs may be related to RNA as the first nucleic acid, a popular idea in evolution.
Exon-shuffling: a model for gene evolution
Some proteins fold into connected, functional sections called domains; these correspond to exons
Perhaps exons were copied, shuffled to create new genes. Several human genes share exons.

27. Relationship between protein domains and exons