Handout for Lecture 2 - C3032
Information You Should Know: DNA to Protein
I. Books for enrichment and background
A. Double Helix - James Watson
B. Girls, Genes, and Gamow - James Watson
C. Eighth Day of Creation - Horace Freeland Judson
D. What Mad Pursuit - Francis Crick
E. Molecular Genetics, 2nd edition - Stent and Calendar
II. Experiments demonstrating the importance of DNA (but also that genetic material can be transferred chemically)
A. Griffiths (1928): the demonstration of transformation
1. Used two strains of Streptococcus pneumoniae, a bacterium that normally causes pneumonia and death in mice.
B. Avery, MacLeod, and McCarty (1944) the experiment that should have won a Nobel prize: the isolation of the transforming activity.
a. R (rough colonies) have a cell wall abnormality and are nonlethal
2. Later showed that this could occur in vitro, i.e., the mouse wasn't required.
b. S (Smooth) are lethal within 24 hrs.
c. Basis UDPG dehydrogenase is defective and UDP-glucuronic acid cannot be incorporated into the capsule polysaccharide
d. Inject R + heat killed S and get virulence so R is transformed.
3. Still later cell extract of S was sufficient for transformation.
1. What needed to be mixed with the R strain was the DNA, not the protein, lipid, or polysaccharides from the S strain.
C. Hershey and Chase and the Waring Blender Experiment
a. Many proteases were tried; none prevented transformation
2. Why did some people doubt this experiment?
b. DNase was added; transforming activity disappeared.
a. DNA base structure was boring, only four bases.
b. Proteins were thought to be complicated.
c. Proteins were known to have detectable activity in minuscule amounts and people did not want to commit what Stent and Calendar (Molecular Genetics) call the Willstätter error, i.e., a small but contaminating amount of protein could be the active principle. By 1949 Rollin Hotchkiss had reduced the protein content to 0.02% in these experiments, but could that have been sufficient for activity?
d. Transformation may be physiological, i.e., it had to do with the particular defect. (Later experiments, however, demonstrated transformation with other traits.)
1. They use T2, a bacterial virus (phage) that infects E. coli
2. Phage is made up of proteins and DNA.
3. They labeled protein with 35S (know why) and DNA with 32P (know why).
4. The shearing of the Waring blender knocked off the 35S, but not the 32P, so DNA entered the bacteria and allowed for the production of new phage.
III. Watson Crick Model (1953)
A. Basic structure
1. Parts list (you be familiar with these names and structures)
B. Model: complementarity through antiparallel strands
a. Base: adenine guanine cytosine thymine (uracil replaces adenine in RNA)
2. Chargaff's rules: A+G=C+T purines=pyridines, A=T & G=C
b. Nucleoside (with the sugar deoxyribose): adenosine guanosine cytidine thymidine (uridine)
c. Nucleotides (nucleosides with phosphate)
3. X-ray structure of fiber- Maurice Wilkins and Rosalind Franklin
a. X-like pattern demonstrated that the molecule was helical.
b. 34Å repeat/10 nucleotides (E. coli 4.2. Mb = 2m)
c. 20Å diameter cylinder
1. Density requires two strands
C. The angle that the sugars form with the bases gives rise to a larger space (the major groove) and a smaller space (minor groove) along the helix
3. H-bonds - You should know how the bases form these bonds with each other.
4. Stacking - van der Waal's interactions (excludes water)
5. Charge consideration: charges from phosphates have to be on the outside.
D. Strands are antiparallel
E. 5' vs. 3'
IV. Meselson/Stahl Experiment - Support for semiconservative replication
A. Made heavy molecules in E. coli with 15N and then switched bacteria to media with 14N
B. Isolated DNA and used CsCl centrifugation to separate molecules of different density.
V. Transcription (DNA -> RNA)
A. Differences between RNA and DNA
1. Uracil instead of Thymine (other modified bases known)
B. Information to review
2. Ribose - chemical consequences: RNA is susceptible to alkaline hydrolysis
1. Types of RNAs (rRNA, tRNA, mRNA, small RNAs)
C. RNA polymerase in E. coli (not used to make primer) complete enzyme (holoenzyme) 480 kd is made of
2. Direction of RNA synthesis (covered in the problems)
3. Note differences between the coding strand and template strand of DNA.
1. A core enzyme [containing 2α 40 kd, β (rif gene) 155 kd, and β' (binds DNA) 160 kd]. The core is basic (so it will bind DNA), but it does not bind to specific sequences in DNA
D. in vitro transcription systems are available for both prokarotes and eukaryotes and have permitted the identification of components needed for transcription and its control.
2. σ (sigma factor), a 85 kd protein
a. Needed for initiation at correct sites by reducing general binding and increasing specific binding.
b. Region it binds is called the promoter.
c. Different σ's allow for different genes to be transcribed.
d. After binding σ comes off so RNA pol core stays bound - winding/rewinding - polymerization.
E. Eukaryotes RNA and polymerases
1.3 RNA polymerases
a. I rRNA
2. RNA has a 7MeG-cap and poly A tail
b. II mRNA α-amanitin-sensitive
c. III tRNA
3. Mature mRNA is often made from the initial transcript by splicing (you should know the difference between introns and exons)
1. Ribosomes (bacterial) - rRNA 60% of mass
B. Direction of synthesis: N to C.
a. 50S proteins - 23S, 5S / 28, 5.8, 5
2. Peptidyl transferase - an RNA enzyme that is part of the ribosome
b. 30S 21 proteins 16S / 33 p 18S
3. Look up the general sequence and structure of tRNAs
a. Usually ~75 nucleotides long
4. You should be familiar with the following terms
b. aa add to to 3' ACC by amino acyl transferases
(1) ATP + aa aminoacyl transferase -> AMP-OOCaa
c. You should know what the anticodon loop is and the direction of the anticodon (e.g., If the codon for Arg is 5'CGA, what is the sequence for the anticodon going 5' -> 3'? ANS: UCG).
(2) AMP-aa + tRNA -> aa-tRNA
a. Initiation factors (IF)
b. Elongation factors (EF)
c. (A and P sites)
d. Release factors (RF)
C. In vitro translation systems are available. The most common eukaryotic systems are from wheat germ and rabbit reticulocytes.