BIOL 231 Exam 2 (11-19)

What is the key takeaway from Fred Griffith’s experiment in the 1920s?

He used S.pneumoniae, which has two strians: a virulent S-strain and a benign R-strain. When het-treating the S-strain and injecting it into a mouse along with healthy R-strain, the mouse died and the bacteria extracted was healthy S-strain. This indicated that something transforms the harmless bacteria into the lethal version.

What is the key takeaway from Avery, MacCleod, and McCarthy (1944)?

They took S.pneumoniae’s S-strain and divided it into RNA, proteins, DNA, lipids, and carbs, then paired each with healthy R-strain. Only the DNA + R-strain produced healthy S-strain, indicating that DNA is the transforming factor.

What did Hrshey and Chase (1950) give concrete, undeniable evidence for?

That DNA contains genetic info.

What is the key takeaway of the Hershey and Chase (1950) experiment?

Using ³²P to mark DNA and ³⁵S to mark proteins, they infected E.coli using viruses with these radioactive markers and blended the mixture to shear off the virus heads. The pellets that were left only had ³²P, indicating that only the DNA caused more viruses.

What is the basic DNA structure?

It is a double helix with a 2nm diameter; each base pair is 0.34nm long, and each turn is 3.4nm, indicating that there are 10 base pairs (bp) per turn.

Describe the backbone of DNA.

It is a deoxyribose sugar with a Phosphate repeated over and over. It has a major and minor groove; the major groove may be easier to access…

Also 3’ to 5’ binding.

Describe the bases of DNA.

They are perpendicular to the long axis; they face in and are tilted 90 degrees, much like a spiral staircase.

Purines (A+G) bind to pyrimidines (T+C+U); A and T bind with 2 H-bonds, and G and C bind with 3 H-bonds.

Describe the polarity of DNA strands.

They have a 5’ and 3’ end; when they come together, they are ANTIparallel.

What is a general rule for transcription and translation?

The template is READ 3-5 and the product is MADE 5-3.

What is chromatin? Describe it.

It is a higher order structure made up of DNA and histones. This is because there’s 3.2 billion bps of DNA in each cell. Histones wind 147 DNA bps to their octomer structure, making a nucleosome, the fundamental unit of chromatin; they come together and coil in multiple orders to make parts of the chromosome.

Describe histones.

They are DNA-binding proteins made up of 8 subunits. They are positively charged and bind to 147 bps of DNA that wrap around twice.

How does chromatin shift in interphase?

Dense chromatin is called heterochromatin. When it diffuses, it becomes euchromatin, which is in active transcription.

What’s the basis of DNA replication?

By binding the OH of a 3’ to the 5’ Phosphate, DNA can be read 3-5 and synthesized 5-3. The DNA being replicated is called the template/parental/old strain, and the DNA being made is called the new strain.

What is meant by semiconservative replication?

When DNA replicates, each of the two double helices contain one intact old strand and 1 new strand.

1/2ⁿ is the equation to calculate what proportion of the DNA is the original template, with n = numbers of replication.

How does the origin of replication work?

DNA is split and replicated here. Each individual split is called a replication bubble, and these grow as DNA is replicated. The ends of each bubble are called replication forks.

What are some issues with DNA replication?

For each strand, there is the lagging strand and the leading strand. The machinery behind replication cannot work backwards.

Single-stranded DNA cannot be a template, as DNA polymerase needs an OH to start.

What is DNA polymerase?

A DNA-dependent DNA polymerase that replicates DNA from an OH.

How are the problems of both the lagging strand and the initiation of replication solved by our bodies?

An RNA primer (DNA dependent RNA polymerase) comes in to start DNA replication; DNA polymerase extends DNA from this primer. Then, Nuclease digests the primer, and repair DNA polymerase fills in the gap. Finally DNA ligase fills in the 1-bp gap.

These bits of replicating DNA on the lagging strand are called Okazaki fragments.

This does NOT work for the very first RNA primer at the end of the DNA molecule.

Why is unwinding the DNA helix an issue?

First you have to open the helix then keep it open. Additionally, you have to prevent the upstream DNA from supercoiling.

How does the body open up and prevent supercoiling of DNA?

DNA Helicase goes along the strand to open it up; single-strand binding proteins prevent the DNA from closing back up; and Topoisomerase cuts a strand and covalently holds on to it to release tension, then ligates the two ends back together.

What is the end problem?

At the 3’ end of the template strand and the 5’ end of the new strand at the LAGGING strand, there is a gap left by the RNA primer. This needs to get filled, as single-strand DNA is not stable and degrades.

How is the end problem solved?

The template strand is elongated via Telomerase and normal DNA synthesis is repeated. The extension is then removed, leaving the template strand degraded.

Why can we solve the end problem by extending the end of the template strand?

The ends of DNA have a TTAGGA repeat, also known as Telomeres. Telomerase takes advantage of these and extends the old DNA.

How does mRNA differ from DNA?

Its sugar has a hydroxyl at the 2’ C, and its bases include uracil instead of thymine.

What are the basics of transcription?

mRNA is made via a DNA-dependent RNA polymerase that READS 3-5 and SYNTHESIZES 5-3. It can open DNA on its own and start independently; however, it only copies one strand.

How does transcription start and stop?

The RNA polymerase scans the dsDNA to find a promoter called a TATA box, which is about -10 bp from the start. Additionally, there is a sequence at -35 bp that signals the polymerase. The polymerase translates until it finds a terminator sequence (TTAA?).

What are the two aspects of mRNA processing?

The 5’ Cap and Polyadenylation.

Describe the 5’ Cap.

It consists of a 7 methyl guanosine and is a 5-5 linkage with three phosphates. This is a ribosome-binding motif, which allows ribosomes to recognize the mRNA.

Describe Polyadenylation.

It is a poly-A tail of about 150-250 bps. It helps with mRNA stability.

How is genetic code degenerative?

It takes three base pairs to make one amino acid.

Describe the basics of codons.

They are in the RNA language (CAUG); they are base triplets read 5-3.

Codons for the same AA are similar, and codons for similar AA (by properties) are also similar. Additionally, some AAs are more than one codon.

What are the start and stop codons?

AUG is methionine, the start codon. Methionine has a Sulfur group.

UGA, UAG, and UAA and the stop codons. These do NOT add anything to the polypeptide chain.

How were codons discovered (is this true?)?

Scientists combined synthetic RNA, cell extract, and radioactively labeled AAs; this yielded proteins with the same radioactive marker.

Describe the basics of the open readin frame.

Every mRNA has three possible reading frames. The OPF starts with the first AUG going 5-3 and ends with the first stop codon in the frame.

What are the five ways changing one base pair can affect a protein?

AA is the same

AA is similar (similar chemistry)

AA is different (nonpolar-polar)

Produce a stop codon

Remove a stop codon

What are tRNAs?

They are transfer RNAs; they are responsible for reading mRNA and matching an AA to it.

It does this by matching its anticodon (going 3-5 with respect to mRNA’s 5-3) with the mRNA.

The AA attaches to the 3’ hydroxyl end of the tRNA.

What is wobble pairing?

It’s the pairing of the third position of the codon not being exactly C:G and A:U; the pairing for the first and second codon is still perfect.

This results in a tRNA pairing with more than one codon.

What are the rules of wobble pairing?

G can go with U, but Inosine (I; G with the amine cleaved off) can pair with anything EXCEPT a G.

What are the basic structural characteristics of the ribosome?

It is composed of both proteins and RNA; the latter of which is responsible for the catalytic activity of the enzyme. It has two subunits.

The large SU has 49 proteins and 3 RNAs, and the small SU has 30 proteins a 1 RNA.

Describe the three sites in the ribosome complex.

The aminoacyl (far right when looking at a 5———-3 strain) site is where a tRNA charged with an AA attaches.

The peptidyl site contains the tRNA with the growing polypeptide.

The exit site has a tRNA with nothing attached.

EPA

5——-3

How does translation initiation work?

The small SU engages with an initiator tRNA (has MET!!!) and accessory proteins. It recognizes the mRNA’s 5’ cap and binds. It then scans for the first AUG; once found, the initiator factors leave, and the large SU binds.

How does translation elongation work?

The growing peptide is in the P site. A random charged tRNA enters the A site; if correct, elongation factor TU (GTP) is utilized to position the tRNA correctly. Peptidyl transferase activity breaks the bond between the polypeptide and tRNA in the P site and makes a new peptide bonds with the tRNA in the A site.

The long SU shifts, then the short SU shifts using elongation factor G. This process continues, and empty tRNAs spit out the E site.

How does translation termination work?

When a stop codon is reached, release factor (RF) binds. It uses hydrolysis of H₂O to break the peptide bond and release the AA chain. Finally, the SUs separate.

What is meant by the concept of a polysome?

Many ribosomes read mRNA simultaneously, much like an assembly line.

What is the only coding RNA?

mRNA. ALL OTHER RNAS are non-coding.

What are two non-coding RNAs important for the regulation of gene expression?

micro RNA (miRNA) and short interfering RNA (siRNA).

Describe the structure and function of miRNAs.

They are post-transcriptional, small (22 nts) RNAs that bind with imperfect complementarity to the 3] untranslated region (UTR) of target mRNAs and destroy them. A single miRNA regulates more than one mRNA. They allow quick response to the environment.

What does miRNA collaborate with to carry out its function?

It brings a protein complex called an RNA induced silencing complex (RISC) to make the miRISC complex; this complex causes the mRNA to be decapped and deadenylated.

How are miRNA and miRISC made?

A precursor miRNA is cleaved by nucleases into uplex miRNA; the duplex splits, and a single strand combines with RISC to make miRISC.

What is the primary function of siRNA?

To respond to foreign strands of RNA by binding with 100% complementarity and lead to cleavage of the foreign RNA.

Describe the biogenesis of siRNA.

Double stranded (ds)RNA is cleaved by nuclease into 22nt segments; a ssRNA segment combines with RISC to make siRISC.

Describe RITS and its role in transcriptional control.

The cleaved dsRNA from siRNA biogenesis makes its way as RNA induced transcriptional signaling (RITS) (is a complex now!) into the nucleus and binds to DNA as nasent RNA. This recruits other proteins and modifies the DNA to shut down transcription.

How can the nucleosome be manipulated to induce transcriptional control?

By remodeling the chromatin in an ATP-dependent fashion.

Phosphorylation, methylation, and acetylation of lysine (acetylated K leads to more transcription) of the histone tails.

Differentiate and describe the long-chain lipids that make up the Pmb.

Phospholipids: polar head group, Phosphate, glycerol backbone, and fatty acid tails.

Sphingolipids: have a serine backbone

Glycolipids: sugar head group; only on the outer membrane

Which long-chain lipid is most prominent?

Phosphatidyl serine (PS).

What are the two characteristics of Lipid Asymmetry, and how is each maintained?

Synthesis: it occurs on the cytosol side of the smooth ER, so the cell uses scramblase to non-specifically move lipids to equal out the distribution between leaflets.

Active sorting: flippase uses specific sorting (PC and glycolipids on outside, PS PE PI on inside) to keep different phospholipids between the two leaflets.

Describe cholesterol and its contribution to the mb.

It has a rigid ring structure (4 rings), an 8C tail, and a small polar head. This allows it to flip and influence mb curvature.

It acts as a buffer for high and low temps, preventing crystallization when cold and excessive fluidity when hot.

How does lipid mobility contribute to the membrane’s considerable mobility?

Phospholipids and particles flex, rotate, and diffuse easily and rapidly many times a second. They also flip, but at a slower rate of daily.

What are two ways to describe phase transition and PT temperature? Graph these ways.

Fluidity and energy absorption. Fluidity should be a population curve, and EA should be a sharp hill.

What are the three ways fatty acids influence PTT?

Pure populations cause sharp transitions.

Longer FA tails have a higher PTT; shorter for lower.

A low # of C=C bonds yields high PTT; high for low PTT.

Cholesterol buffers the effect of high and low temp.

What are the three types of membrane proteins?

Integral, peripheral, and lipid-linked.

Describe the fundamental characteristics of integral mb proteins.

They fully span the mb at least once with a span of H-phobic R groups. The mb must be dissolved to remove them.

Describe the fundamental characteristics of peripheral mb proteins.

They form non-covalent i/a with mb by associating with lipids or proteins. They can be extracted with salt.

Describe the fundamental characteristics of lipid-linked mb proteins.

They have covalent i/a with lipids. These are reversible and are important for signaling.

What did Fry and Edidin’s experiment show about protein mobility?

After fusing a mouse and human cell, each with their labeled integral proteins, the proteins diffused throughout the membrane and cell as time passed.

What did fluorescence recover after photobleaching (FRAP) tell us?

When marking proteins and then bleaching a certain area, you can track how the labeled proteins diffuse randomly and gradually restore fluorescence to the bleached patch.

What is the purpose of single particle tracking?

To quantify the diffusion coefficient of a particle by tracking its movement.

How do secondary structures play into integral mb proteins?

If it’s an alpha helix, it spans the mb; these typically are 20-30 AA h-phobic residues.

Beta sheets have sides with differing polarity and form beta barrels.

What are the five functions of membrane proteins?

Receptors, transporters (active/passive), enzymes, regulators, and junctions.

A single protein can do more than one of these!!!

Describe nerve growth factor receptor (NGFR)’s structure and function.

It is a transmembrane protein that spans the mb once with a singular alpha helix. It is a receptor on the outside and an enzyme on the inside with protein kinase phosphorylating tyrosine.

What do kinases do?

They transfer phosphorus to a substrate protein.

What is the process of NGFR and kinase activity?

NGF (a dimer) binds to NGFR, inducing its dimerization; this triggers tyrosine kinase activity as the NGFRs come together and interact at their domains, phosphorylating each other. This creates binding sites for other proteins via free P, which proteins take advantage of and assemble on the NGFR.

What is a common trait among ion channels?

They allow diffusion down an ion’s [ ] gradient.

Draw the Sodium Channel and a sequential aerial view of one segment.

It should have four repeats of S1-6 (crosses of the membrane) with a P region between S5 and 6 that does NOT fully cross the membrane. The N and C terminus should be on the inside.

The final aerial view should look like a four-leaf clover with the P domains facing in.

Describe the Na⁺/Glucose symporter.

It uses the energy of the [Na⁺] gradient to move glucose; this occurs in regions where [Na_o] > [Na_i] and [gluc_o] < [gluc_i].

The two binding sites allow for cooperativity between Na and glucose; the symporter binds to Na with every glucose. The binding of Na increases affinity for glucose.