1. G3 course reading - Tantin
MBIOL 6420
8:30-9:30 ASB 210
Dean Tantin, PhD
X7-3035,
Required course reading
For many of you, this is a review of undergraduate material, but study these slides in advance of the first day of my lectures. There is much that is testable in here, and things in the next few lectures will rely on knowing this material.

2. (Simplified) flow of genetic information…
translation
splicing
transcription
DNA
mRNA
Protein
pre-mRNA
The central dogma
Francis Crick

3. Gene expression begins with the process of TRANSCRIPTION
Some terminology (applies to prok. and euk. transcription):
Transcription is the synthesis of an RNA chain from a DNA template.
RNA synthesis proceeds in the 5’3’ direction and is performed by RNA polymerase and ancillary factors.
RNA synthesis utilizes NTPs, with uridine triphosphate (UTP) in place of dTTP in base pairing with adenosine. Thus, RNA is marked by the presence of a 2’ hydroxyl and uridylate.
Only one strand of the DNA double helix is utilized as the physical template for RNA synthesis, and is termed the template strand.
The coding strand has a 5’-3’ sequence that is identical to the 5’- 3’ sequence of the RNA (with U in place of T).

4. Gene expression begins with the process of TRANSCRIPTION
More terminology:
The promoter is a region of DNA at the 5’ end of the gene that controls its expression. In eukaryotes it includes the initiation site (start site), the first base that is transcribed (in bacteria this can overlap with a negative element termed the “operator”).
The terminator is a DNA sequence that directs where RNA synthesis stops (this is different from a stop codon). The intervening DNA is considered the “body” of the gene. A transcription unit is all RNA synthesized in a single RNA chain. An “operon” is a bacterial transcription unit that consists of multiple open reading frames coding for multiple proteins.
The standard nomenclature for describing the position of DNA base pairs relative to the start site considers +1 as the start site and -1 as the base before it (there is no zero). Downstream DNA is any DNA that is found in the direction the RNA polymerase travels. The opposite is true for upstream DNA.

5. Gene expression begins with the process of TRANSCRIPTION

6. The basic form of the cycle is conserved in eukaryotes.
Overview of the prokaryotic transcription cycle
The basic form of the cycle is conserved in eukaryotes.
Promoter (template) recognition:
Core RNA polymerase cannot find a promoter by itself. It requires promoter specificity factors, called sigma (s) factors.
Open complex formation and initiation:
--The DNA is unwound to form the bubble.
--Formation of the first phopshodiester bonds.
2-9 base “abortive transcripts” synthesized.
Promoter clearance (promoter escape):
The transition to processive transcription; the bubble travels with the polymerase.
Elongation:
Incorporation of ribonucleotides into the RNA chain.
Termination:
Synthesis ends and the RNA is released.
The enzyme first binds the promoter. The DNA strands are then separated (“melted”) by the polymerase (as is the case for most bacterial transcription), or by the action of polymerase plus the help of ancillary factors (as is the case for eukaryotic Pol II transcription). The structure that contains melted DNA and polymerase is called the “open complex”.

7. Promoter recognition—bacterial RNA polymerase
Two forms:
‘Core’ with the subunit composition aabb’w (=a2bb’w).
‘Holoenzyme’* also includes s.
There are multiple s factors, so the polymerase can recognize promoters with different recognition sequences.
The most common s factor is s70.
Eukaryotes lack true sigma factors, and have different strategies for recognizing promoters.
*‘Holoenzyme’ refers to an active form in which all the subunits and cofactors needed for activity are present (as contrasted with ‘apoenzyme’).
Beta prime is the largest subunit. As we will see, the core subunits have homologs in each of the three eukaryotic RNA polymerases. The two alpha subunits are involved in enzyme assembly and promoter recognition. They bind some bacterial activators. The beta and beta prime subunits form the catalytic center, and the sigma subunit provides promoter specificity. The full E. coli enzyme is approximately 465 kDa.

8. Promoter recognition—bacterial RNA polymerase
b’—largest subunit. Forms part of active site. Interacts nonspecifically with DNA and nascent RNA.
b—forms the rest of the active site. Interacts nonspecifically with DNA and nascent RNA.
a—determinants for assembly of RNAP. Recognizes DNA nonspecifically, OR can recognize a specific Upstream Promoter element (“UP element”) at certain highly expressed bacterial genes.
w—stabilizes the assembled RNAP.
Sigma factors greatly increase the affinity of polymerase for promoter DNA (by decreasing the off rate). Holoenzyme bound to DNA that is unmelted is called the closed complex.
Beta prime is the largest subunit. As we will see, the core subunits have homologs in each of the three eukaryotic RNA polymerases. The two alpha subunits are involved in enzyme assembly and promoter recognition. They bind some bacterial activators. The beta and beta prime subunits form the catalytic center, and the sigma subunit provides promoter specificity. The full E. coli enzyme is approximately 465 kDa.

9. Transcription is a HEAVILY REGULATED process
Transcription is frequently regulated both temporally (e.g., response to signals, stress) and spatially (i.e., particular DNA segments transcribed).
Regulation largely but not entirely occurs prior to transcription complex assembly and active phosphodiester bond formation.
This means that the recognition needed for regulation must be of standard unmelted B-form DNA (or of chromatin, which uses the underlying DNA sequence to organize itself). Either way, DNA: see attached review from Coller and Kruglyak (“It’s the Sequence, Stupid!”)
Following this line of reasoning, cells must use strategies to recognize sequences in unmelted DNA to direct when and where the transcription machinery is active.

10. The DNA helix and how it is read
Yellow: zinc finger protein recognizing DNA…
22Å
12Å
34Å
22Å

11. The DNA helix and how it is read
Specific chemical groups exposed in the major and minor grooves provide information about the DNA sequence.
The sequence is“read” through specific chemical interactions (e.g., hydrogen bonding) with transcription factors that fit into these grooves and can only recognize the chemical groups in a specific pattern.

12. “Wiring diagrams” provide a shorthand for regulation