Molecular Biology Midterm

Lectures 2-9

Nucleotide is made up of?

A nitrogenous base + sugar + phosphate group(s)

DNA Sugar:

Deoxyribose

RNA Sugar:

Ribose

What are the DNA Bases:

adenine, thymine, cytosine, guanine.

What are the RNA Bases:

adenine, uracil, cytosine, guanine.

DNA Base Pairings:

A pairs with T; G pairs with C.

RNA Base Pairings:

A pairs with U; G pairs with C.

How many hydrogen bonds between A-T?

2 hydrogen bonds.

How many hydrogen bonds between G-C?

3 hydrogen bonds.

Which DNA is more stable, GC-rich or AT-rich

GC-rich because G-C has 3 hydrogen bonds.

5' and 3' ends:

The directionality of a nucleic acid strand. New DNA/RNA is synthesized 5' → 3'.

Antiparallel

The two DNA strands run in opposite directions, one 5' → 3' and the other 3' → 5'.

DNA polymerase synthesis direction

5’—> 3’

What does DNA polymerase need to begin synthesis?

A primer with a free 3’ OH group

Leading strand:

Synthesized continuously

Lagging strand:

Synthesized discontinuously as Okazaki fragments

What are Okazaki Fragments?

Short DNA fragments made on the lagging strand

DNA polymerase proofreading:

Removes incorrectly incorporated nucleotides to improve accuracy.

DNA polymerase processivity:

The ability of DNA polymerase to add many nucleotides without falling off the DNA

What is the central dogma?

DNA—> RNA—> Protein

Transcription:

DNA—> RNA

Translation:

RNA—> Protein

mRNA:

Carries the genetic information used to make a protein.

tRNA:

Brings amino acids to the ribosome during translation.

rRNA:

Major structural/catalytic component of the ribosome.

RNA polymerase:

Enzyme that makes RNA during transcription

Does RNA polymerase need a primer?

No

Promoter:

DNA sequence where the transcription machinery assembles to begin transcription

Enhancer:

Regulatory DNA sequence that can increase transcription

Transcription Factors:

Proteins that regulate transcription by binding DNA and/or interacting with transcription machinery

TATA Box:

A promoter associated DNA sequence involved in transcription initiation

Where is eukaryotic transcription located?

The nucleus

Where is eukaryotic translation located?

Cytoplasm/riibosomes

Can prokaryotes couple transcription and translation?

yes

Can eukaryotes couple transcription and translation?

No, because transcription occurs in the nucleus and translation occurs in the cytoplasm.

5’ cap:

Modification added to the 5’ end of the eukaryotic mRNA.

Poly-A-Tail:

Adenine rich sequence added to the 3’ end of the eukaryotic mRNA.

If given 5'-ATGC-3', what is the complementary strand?

3'-TACG-5'.

If asked to write that complementary strand 5' → 3(5'-ATGC-3')

5'-GCAT-3'.

What must you remember when writing a complementary DNA strand?

Base pairing AND directionality. The strands are antiparallel.

5'-ATGCCAT-3' → complementary strand written 5' → 3'?

5'-ATGGCAT-3'

PCR:

Polymerase Chain Reaction; amplifies a specific DNA sequence

What does PCR require?

Template DNA, forward primer, reverse primer, thermostable DNA polymerase, dNTPs, and buffer.

PCR denaturation:

DNA strands separate at high temperature

Typical PCR denaturation temperature:

About 94–98°C.

PCR annealing:

Primers bind to complementary sequences on the template DNA.

Typical PCR annealing temperature:

About 50–65°C.

PCR extension:

DNA polymerase extends the primers and makes new DNA.

Typical PCR extension temperature for Taq:

About 72°C.

Taq polymerase:

Thermostable DNA polymerase commonly used in PCR

Why is Taq thermostable?

It can survive the repeated high temperatures used during PCR.

Why are primers necessary in PCR?

They provide a starting 3' OH for DNA polymerase and determine what region is amplified.

Amplicon:

DNA product amplified by PCR.

Typical PCR cycles:

About 25–35 cycles.

PCR amplification:

The amount of target DNA theoretically doubles each cycle.

High-fidelity PCR polymerase:

A polymerase with proofreading ability and lower error rates than Taq.

What happens if there is a mismatch at the 3' end of a primer?

It can strongly interfere with extension/PCR efficiency.

DNA melting:

Separation of double-stranded DNA into single strands by breaking hydrogen bonds.

Tm:

Melting temperature; the temperature at which DNA strands begin to separate significantly.

Main factor affecting Tm tested in your quizzes:

GC content.

Higher GC content does what to Tm?

Increases Tm.

Why does GC-rich DNA have a higher Tm?

G-C pairs have 3 hydrogen bonds.

What happens above Tm?

DNA strands separate.

What happens below Tm?

DNA strands are more likely to remain paired.

What does a mismatch do to Tm?

Decreases Tm.

What generally happens to Tm as DNA length increases?

Longer DNA generally has a higher Tm.

qPCR:

Quantitative PCR; measures DNA amplification in real time using fluorescence.

SYBR Green:

Fluorescent dye used to monitor double-stranded DNA in qPCR.

Ct:

Cycle threshold; the cycle at which fluorescence reaches a detectable threshold.

Low Ct means:

More starting target DNA.

High Ct means:

Less starting target DNA was present.

Why does a sample with more starting DNA have a lower Ct?

It reaches the detectable fluorescence threshold in fewer cycles.

Why does a sample with more starting DNA have a lower Ct?

It reaches the detectable fluorescence threshold in fewer cycles.

What does qPCR allow you to measure?

The amount of target DNA and changes in gene expression.

The amount of target DNA and changes in gene expression.

The amount of amplified DNA being produced.

Major advantage of qPCR:

It allows real-time quantification of DNA amplification.

Basic structure of an amino acid:

Amino group + carboxyl group + hydrogen + R group attached to a central carbon.

R group:

Variable side chain that determines an amino acid's unique properties.

What determines an amino acid's chemical properties?

Its R group.

Cysteine:

Contains sulfur and can form disulfide bonds.

Glycine:

Smallest amino acid.

Proline:

Has a ring structure that restricts the backbone and can disrupt α-helices

Methionine:

Usually the first amino acid incorporated during translation.

N-terminus:

End of a polypeptide with the free amino group

C-terminus:

End of a polypeptide with the free carboxyl group.

Peptide bond:

Covalent bond connecting amino acids.

How is a peptide bond formed?

Between the amino and carboxyl groups of amino acids, releasing water.

Polypeptide:

Chain of amino acids connected by peptide bonds.

Primary protein structure:

The amino acid sequence of a protein

Why is primary structure important?

The amino acid sequence helps determine the protein's final 3D structure and function.

First amino acid in most eukaryotic proteins:

Methionine.

Secondary protein structure:

Local folding of a polypeptide into α-helices and β-sheets.

What stabilizes α-helices and β-sheets?

Hydrogen bonds between backbone atoms.

α-helix:

Coiled secondary structure stabilized by backbone hydrogen bonds.

How many amino acids are in one turn of an α-helix?

About 3.6 amino acids

Which amino acid is least likely to occur in an α-helix?

proline

Why does proline disrupt α-helices?

Its ring structure restricts the backbone.

β-sheet:

Secondary structure formed by β-strands connected by hydrogen bonds.

β-sheets can be:

parallel or antiparallel

Where are the hydrogen bonds in a β-sheet?

between neighboring β- strands