1. BIOL 2416 CH 5: Transcription

2. DNA (1 gene). pre-mRNA (a. k. a. primary transcript). mature mRNA
DNA (1 gene) pre-mRNA (a.k.a. primary transcript) mature mRNA protein (1 polypeptide)
Eukaryotic Gene Expression
transCription in nucleus
by RNA Polymerase
RNA processing in nucleus
transLation in cytosol
by ribosomes

3. Gene expression has NOTHING to do with DNA Replication
DNA Replication occurs just before cell division (mitosis or meiosis)
It’s about making more DNA
Gene expression occurs in NON-dividing cells
It’s about making RNA (transcription) and protein (translation)

4. DNA replication
transcription
Make what from what?
DNA copy from DNA
RNA from DNA
# template strands 2 1
Primer needed?
Yes no
Precursors
dNTPs (GATC)
NTPs (GAUC)
Main enzyme
DNA Polymerases
RNA Polymerases
Bi or uni?
Bi-directional; growing bubble
Uni-directional; moving bubble

5. Transcription = RNA production from a DNA template:
Nontemplate DNA strand: 5’GATCGATC3’ Template DNA strand: 3’CTAGCTAG5’ transcription RNA strand: 5’GAUCGAUC3’

6. Kinds of RNAs (for now…)
mRNA
tRNA
rRNA
snRNA
prefix
messenger
transfer
riboso-mal
small nuclear
function
encodes amino
acid sequence of protein
brings amino acids to ribosomes during translation
catalytic part of ribosome
part of spliceo-some (eukarytic RNA processing)

7. What does a prokaryotic gene look like?
(prokaryotic promoters)
(TTGACA) (TATAAT); a.k.a. Pribnow Box

8. Transcription Mechanism
Initiation
Elongation
(highly conserved between prokarytotes and eukaryotes
Termination

9. Prokaryotic transcription initiation:
RNA Polymerase holoenzyme (= core of two a and two b’ subunits, plus a s (“sigma”) factor) loosely binds -35 promoter sequence, then tightly to -10 promoter sequence.
(Promoters are built-in DNA sequences that “promote” transcription of the gene that follows)
RNA Polymerase holoenzyme untwists about 17 bp, creating a small bubble.

10. Different sigma (s) factors help prokaryotic RNA Polymerase choose promoters/genes:
Sigma 70 recognizes most abundant garden-variety -35/-10 promoter
Sigma 32 recognizes -39 (CCCCC)/-15 (TATAAATA) promoters of some heat-shock/stress genes
Sigma 23 recognizes -15 (TATAATA) in T4-infected cells
And more…

11. Prokaryotic transcription elongation:
RNA Polymerase holoenzyme begins to synthesize RNA (in 5’ to 3’ direction) by reading the DNA template strand (3’ to 5’).
After the “transcript” consists of 8-9 new RNA nucleotides, sigma factor has done its job (find/bind promoter) and is released.
RNA Polymerase core enzyme continues to add NTPs to the growing RNA strand; DNA helix reforms, displacing the RNA transcript; transcription bubble moves downstream across the gene at ntd/s.

12. Fig Chemical reaction involved in the RNA polymerase-catalyzed synthesis of RNA on a DNA template strand
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

13. Fig. 5.4 Action of E. coli RNA polymerase in the initiation and elongation stages of transcription
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

15. Prokaryotic transcription termination:
Happens at a terminator sequence (2 kinds:)
Rho-independent (Type I) terminators consist of a palindrome that folds up on itself in a hairpin, followed by 4-8 Us in the transcript; this makes the DNA/RNA hybrid fall apart.
Rho-dependent (Type II) terminators don’t have the Us and may not have the hairpin region.  protein binds ATP and RNA ; moves along transcript to destabilize DNA/RNA binding

16. Fig. 5.5 Sequence of a -independent terminator and structure of the terminated RNA
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

18. Eukaryotic RNA Polymerases
Location
Product
-amanitin sensitivity
RNA Pol I
Nucleolus
28S, 18S, 5.8S rRNAs
insensitive
RNA Pol II
Nucleoplasm
mRNAs and some snRNA
very sensitive
RNA Pol III
tRNAs, 5S rRNA and rest of snRNAs
Less sensitive

19. Eukaryotic RNA Polymerase II promoter elements:
Core promoter elements ( within -50)
-25/TATAAAA (“TATA box”); aids local denaturation and sets start point for transcription
Initiator (Inr) element; pyrimidine-rich sequence near txn start also sets start point
Promoter-proximal elements (-50 to -200)
-75/CAAT box for promoter efficiency
-90/GGGCGG (“GC box”) for promoter efficiency

20. Enhancer sequences (as far as +/- 3000)
Eukaryotic promoter = combination of core and promoter-proximal sequences
Enhancer sequences (as far as +/- 3000)
Boost transcriptional rates from promoter
Eukaryotic transcription involves the assembly of many different proteins to bind to promoter sequences
Different combinations of proteins work with different promoters
Each eukaryotic gene is under the control of its OWN promoter (producing monocistronic mRNAs)
Combinatorial way to control which genes get transcribed and which do not

21. All 3 eukaryotic RNA Polymerases need own transcription factors
Needed for initiation; important for control of gene expression
Abbreviated “TF”
Numbered according to RNA Polymerase number (specific for one RNA Pol)
Lettered in order of discovery

22. Transcription by eukaryotic RNA Pol II requires a complete PIC (Pre-Initiation Complex):
TFIID binds TATA box
TFIIB binds TFIID-TATA complex
TFIID-TATA-TFIIB recruits RNA Pol II and TFIIF
TFIIE and TFIIH also bind
Complete PIC provides basal (low) levels of transcription

23. For cell-specific gene expression, low PIC activity is modulated by:
Activators binding enhancers (located upstream, within gene, or downstream; close or far away; in either orientation) to boost transcription rates.
Repressors binding silencers to decrease transcription rates (not as common).

24. Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

25. RNA processing of eukaryotic primary transcripts, a.k.a. pre-mRNA:
5’ capping (addition of m7Guanosine) to aid ribosome binding
3’ poly(A) tailing for (eu) transcript stability and (eu)transcription termination
Splicing: removal of introns; cutting and
pasting together of exons
(NONE of the above is seen in prokaryotes!)

26. Fig. 5.11 General sequence of steps in the formation of eukaryotic mRNA
Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

27. Splicing is done
with a
spliceosome
complex:
Exons glued together

28. RNA Editing
Post-transcriptional insertion or deletion of RNA nucleotides, or
Post-transcriptional conversion of one RNA base into another
Can involve as much as 50% of mature mRNA