Topic 2 Flashcards
Structure of Adenine
- C6 amino group
Structure of Guanine
- C6 oxy (C=O) group
- C2 amino group
Structure of Cytosine
- C4 amino group
- C2 oxy group
Structure of Thymine
- C2 oxy group
- C4 oxy group
- C5 methyl group
Between the major and minor groove, which groove is deeper?
Major
Size and diameter of minor groove
12 Å, 1.2 nm
Size and diameter of major groove
22 Å, 2.2 nm
From where are the angles measured for the major and minor grooves?
The glycosidic bonds
The DNA sequence of operator sites that are recognized by the λ repressor and Cro are…
Palindromes
- not perfect palindromes, but very close
- The palindrome sequences are what allows the λ repressor and Cro to bind to the operators
How many peptide bonds can an amino acid form?
2 (one at the amino end, one at the carboxyl end)
In a polypeptide chain, where is the peptide backbone found?
On the inside (in contrast to DNA, where the sugar-phosphate backbone is on the outside)
- R groups protrude outward (in contrast to DNA: bases point inward)
What does the 3D folding of a polypeptide determine about a protein?
The function/activity of a protein
What are the two commonly found secondary structures?
- alpha helix (side chains protrude out)
- beta sheet (side chains project alternatively to either side of the backbone)
Tertiary structure of proteins is found in…
Physiological conditions
Quarternary structure of proteins is…
Composed of multiple polypeptide chains (each peptide chain is called a subunit)
Proteins fold into discrete modules known as…
Domains
In the cI repressor, how many helices are in the N terminus? How are they linked together?
5 helices in the N terminus linked together by a linker sequence.
Which alpha helix in the lambda repressor recognizes and fits into the major groove of the operator DNA?
3
Function of alpha helices 2 in the N domain of the cI repressor?
Important for positioning helix 3
What do helices 2 and 3 form?
A helix-turn-helix (HLH) motif for OR binding
Alpha helix 1 function in N domain of the cI repressor
Also helps position the DNA binding domain of the repressor onto the major groove
The side chains of the λ repressor fit into the…
Major groove on the operator
The lambda repressor is symmetrical. What does this indicate regarding its DNA binding?
Each monomer of the dimer binds to half of the palindromic operator sequence. This means the operator sequence is symmetrical, and the repressor dimer fits perfectly onto it.
How does the lambda repressor enhance binding to the operator?
Repressor arms wrap around the operator to enhance binding
Describe the tertiary structure of Cro
3 alpha helices and 3 beta sheets (rather than 5 alpha helices like lambda repressor)
True or false: Cro binds to the same OR site as the repressor but in a different major groove
False.
- Cro binds to the same OR site as the repressor in the same major groove (which leads to competition for binding)
In general, what is it about the λ regulatory proteins that determine the relative affinity for OR sites?
Their amino acid residues.
What are the conserved amino acid residues in helix 3 of Cro and λ repressor that are involved in base contact with the operators?
Gln and Ser
In the λ repressor and Cro, there are Gln and Ser residues. How does this contribute to DNA binding?
Gln and Ser are both polar, which helps them interact with the DNA (which is also polar)
In the λ repressor, there is an alanine residue. How does this residue contribute to DNA binding?
The alanine is nonpolar, so it’s probably not directly interacting with the DNA, but can create a hydrophic environment which helps DNA and protein aggregate due to the hydrophobic effect.
In the Cro protein, there is a Lys residue. How does this residue contribute to DNA binding?
The Lys residue is charged, which helps the protein interact with the negatively charged DNA.
How does the glutamine in the repressor protein specifically interact with the adenine in the operator DNA?
2 hydrogen bonds form between the side chain of Gln and the base of A in the major groove.
Alanine is found in helix 2 in the λ repressor and in Cro. What purpose does this alanine serve?
It interacts with Val (helix 3 in repressor) and Ile (helix 3 in Cro) through the hydrophobic effect to help maintain the structure of the protein and position helix 3.
Glutamine is found in helix 2 in the λ repressor and in Cro. How does this Gln help in protein binding to the operator?
Gln is polar and interacts with the DNA phosphate backbone through ion-dipole interactions.
What two regions in the RNA polymerase binding site are recognized?
The -10 and -35 regions
What specific protein (transcription factor) recognizes the -10 and -35 regions in the RNA polymerase binding site, and what is its function?
The sigma factor recognizes these regions and recruits RNA pol
The -10 region in the RNA pol binding site is known as _____ in eukaryotes, and _____ in prokaryotes
Eukaryotes: TATA box
Prokaryotes: Pribow box
When the λ repressor binds to OR2, it enhances RNA pol binding to PRM (λ repressor expression) but represses RNA pol binding to PR (Cro expression). How does this happen?
PR has 3 base pairs at its -35 region and PRM has 2 base pairs at its -35 region that overlap with OR2. When the repressor binds to OR2, these base pairs are covered. Since 3 base pairs are covered by the repressor in PR and only 2 in PRM, PRM is still able to bind RNA pol but PR cannot.
How do the residues in the λ repressor contribute to enhanced cI gene expression?
2 amino acid residues along helix 2 and 1 in the loop between helix 2 and helix 3 are responsible for interacting with RNA polymerase to positively promote transcription initiation.
What is the angle associated with the major groove?
210 degrees
What is the angle associated with the minor groove?
150 degrees
How can you determine the base pairing when looking at the major groove?
Look at the order of nitrogens and oxygens exposed to the major groove. If blue, red, blue, it’s GC. If blue, blue, red, it’s AT. If red, blue, blue, it’s TA.
