Mod 5 Flashcards
What is the structure of DNA?
- DNA is double-stranded, with strands made of ATCG.
- The strands are antiparallel: one runs 5’ → 3’ and the other 3’ → 5’.
What is the function of helicase in DNA replication?
Helicase separates the DNA strands, creating the replication fork and providing two template strands for replication.
How does DNA polymerase work in replication?
- DNA polymerase synthesizes new strands in the 5’ → 3’ direction.
- It uses the parental strand as a template to create a complementary new strand.
What is the difference between leading and lagging strands in DNA replication?
Leading strand is synthesized continuously in the 5’ → 3’ direction.
Lagging strand is synthesized discontinuously in Okazaki fragments, requiring multiple primers.
What happens after the primers are used in DNA replication?
- Exonuclease removes the RNA primers.
- DNA polymerase fills in the gaps with DNA.
- DNA ligase seals the strands together to form complete double strands.
Why is DNA replication called semi-conservative?
One strand of the original DNA molecule is conserved in each new DNA molecule.
What is meant by bidirectional replication?
Replication occurs in both directions from the origin of replication along the replication fork.
What is the role of DNA polymerase I?
DNA polymerase I removes RNA primers and replaces them with DNA during replication.
2 types of replication
- Conservative - one of the two daughter duplexes is the conserved parental duplex while the other is synthesized de novo
- Dispersive - parental material is scattered through the structures of the daughter duplexes
What are the key types of DNA polymerase in E. coli?
DNA Poly I: Cleanup function, not for main replication.
DNA Poly II/III: Main enzymes for replication, with Poly III being the main replication enzyme.
DNA Poly IV/V: Involved in DNA repair.
What is the significance of DNA polymerase III?
- DNA polymerase III is the main enzyme for DNA replication in E. coli.
- It has a high processivity, meaning it can catalyze many consecutive reactions without releasing its substrate.
- Uses an RNA primer made by primase
- Works as a dimer, doin leading and lagging strand synthesis at the same time
2 reasons for fidelity of DNA replication
- Active site constraints
DNA poly I active site can only accommodate the correct W-C base pairs - Proofreading activity
Allows them to double-check their work and they have 3’ to 5’ exonuclease activity to fix their mistakes during a kinetic pause
What is the central dogma of molecular biology?
DNA → RNA → Proteins
What are the stages of bacterial transcription?
Initiation: RNA polymerase binds to the promoter sequence.
Elongation: RNA polymerase synthesizes the RNA strand.
Termination: RNA polymerase stops at a termination sequence.
What are the three types of RNA and their functions?
mRNA (messenger RNA): Encodes proteins.
tRNA (transfer RNA): Brings amino acids to ribosomes during translation.
rRNA (ribosomal RNA): Composes ribosomes and facilitates translation.
How is transcription different from DNA replication?
- Transcription uses RNA, not DNA.
- It requires only one strand of DNA as a template.
- Transcription does not require a primer.
What is the role of RNA polymerase in transcription?
- RNA polymerase synthesizes RNA from a DNA template.
- It lacks proofreading ability exonuclease, leading to a higher error rate than DNA replication.
What are the key elements in bacterial promoters for RNA polymerase binding?
-10 region (TATAAT) and -35 region (TTGACA) are consensus sequences.
The UP element (A/T rich) further helps in RNA polymerase binding.
What is the difference between the sense and antisense DNA strands in transcription?
Sense strand: Matches the RNA sequence (except for thymine replaced by uracil).
Antisense strand: Used as the template for RNA synthesis.
How does transcription initiation and elongation occur in bacteria?
- RNA poly directed by bound sigma factor to DNA promoter
NOT DONE
How does transcription termination occur in bacteria?
Rho-independent: A hairpin structure forms, causing RNA polymerase to pause and dissociate.
Rho-dependent: The Rho protein unwinds the RNA-DNA hybrid, causing termination.
What is the role of mRNA in translation?
mRNA carries the genetic code from the DNA and is translated into a protein.
How do ribosomes participate in translation?
Ribosomes read the mRNA codons and assemble the amino acids into proteins.
Ribosome binds to mRNA and tRNAs and other soluble factors required for translation
What is the Shine-Dalgarno sequence?
Untranslated region
Consensus sequence which allows for proper positioning of the start codon (AUG) on the mRNA relative to the ribosome, which allows for initiation of translation
What is the role of tRNA in translation?
tRNA carries amino acids and matches them to codons in the mRNA during translation.
What is the function of aminoacyl-tRNA synthetases?
These enzymes attach the correct amino acid to its corresponding tRNA.
Amino acid carboxylate and the ribose 3’ OH of the 3’ terminal adenosine on the tRNA
How is translation initiated in bacteria?
- The ribosome binds to the mRNA’s Shine-Dalgarno sequence.
- The first tRNA (f-Met) binds to the start codon (AUG).
What are the steps in translation elongation?
- tRNA Binding: tRNA enters the ribosome.
- Peptide Bond Formation: The amino acid from the P-site is transferred to the A-site.
- Translocation: The ribosome moves one codon forward on the mRNA.
* Uncharged tRNA in P-site shifts to E-site and is released.
* A-site tRNA (with additional amino acid) shifts to P-site.
How does translation terminate?
- A stop codon (UAA, UAG, or UGA) enters the A-site.
- A release factor binds, causing the polypeptide to be released.
What is PCR (Polymerase Chain Reaction)?
- PCR is a technique used to amplify or make many copies of a specific region of DNA.
- It is done in vitro, meaning it occurs in a test tube, not in a living organism.
- The main benefit of PCR is that it generates enough copies of a DNA region to be used in other applications such as sequencing, gel electrophoresis, or cloning into a plasmid.
What is Taq polymerase, and why is it important in PCR?
- Taq polymerase is a type of DNA polymerase enzyme that synthesizes new DNA strands using the old strand as a template.
- It is named after Thermus aquaticus, a heat-tolerant bacterium found in hot springs and hydrothermal vents.
- Taq polymerase is heat-stable, unlike other DNA polymerases, and works most efficiently around 70°C, which is essential for PCR’s high-temperature steps.
What are PCR primers and what role do they play in PCR?
- PCR primers are short sequences of nucleotides (usually around 20 bases long) that serve as the starting point for DNA synthesis.
- They are required for all polymerases, including Taq polymerase.
- The primers determine the region of DNA that will be amplified by binding to complementary sequences on opposite strands of the DNA template, flanking the target region.
What are the three main steps of PCR, and what happens during each?
- Denaturation (96°C):
* Heat the DNA to separate the double strands into single strands, providing templates for the next step. - Annealing (55-65°C):
* Cool the DNA so that the primers can bind to their complementary sequences on the single-stranded template DNA. - Extension (72°C):
* Raise the temperature so that Taq polymerase extends the primers, synthesizing new DNA strands.
These steps are repeated 25-35 times, typically taking 2-4 hours
How does PCR result in exponential DNA amplification?
- PCR is an exponential process. After each cycle, the number of DNA copies doubles.
- This exponential growth happens because the newly synthesized DNA strands serve as templates for the next round of amplification.
- As a result, billions of copies of the target DNA region can be generated in just a few hours.
What is gel electrophoresis, and how is it used in PCR?
- Gel electrophoresis is used to visualize the results of PCR.
- DNA fragments are loaded into a gel matrix and subjected to an electric current.
- The DNA fragments move through the gel, with smaller fragments traveling faster than larger ones.
- This process separates the fragments by size, forming bands that represent the amplified DNA regions.
- A DNA ladder is also used to determine the size of the DNA fragments.
What are some common uses of PCR in various fields?
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Genetic testing: For diagnosing genetic disorders and identifying mutations.
Amplification of fetal DNA: For prenatal testing and analysis.
Testing for viruses or pathogens: PCR can detect the presence of specific viral or bacterial DNA.
Forensics: PCR is used to match DNA samples to suspects, such as in criminal investigations.
Amplification of blood or tissue samples: To gather sufficient DNA for further analysis.
What are STRs (Short Tandem Repeats), and how are they used in forensic analysis?
- STRs are short (2-5 nucleotide) sequences of DNA that repeat multiple times in a row.
- Alleles differ based on the number of STR repeats they contain.
- STR analysis can help build a unique genetic fingerprint, useful in forensic science for matching suspects to crime scene evidence.
- STRs are highly polymorphic, meaning they vary greatly among individuals, which makes them valuable for personal identification.
