Micro Exam 2 Flashcards
Define a genome
All genetic material of an organism, including genes that encode proteins, rRNA, tRNA, and small RNAs
Describe the function of a genome
Store and transmit all genetic info necessary for the organism to function, develop, and reproduce
Briefly describe the size and nature (circular) of microbial genomes in comparison to eukaryotic genomes and the human genome
Microbial genomes: typically circular, range from 490-9,400 kb
Eukaryotic genomes: larger, linear, contian more noncoding DNA
Distinguish between monocistronic and polycistronic genes
Monocistronic: one gene, one protein (transcribes individually)
Polycistronic: multiple genes transcribed as a unit (operon)
Define an operon
A set of genes controlled by a single promoter (cluster of genes; 2 or more)
Describe the structure of DNA with respect to its constituent monomers (nucleotides) and its helical structure; what holds the strands together?
Structure:
-Nitrogenous base (ATGC)
-Deoxyribose sugar
-Phosphate group
DNA Strands: antiparallel (5’ –> 3’ and 3’ –> 5’)
Describe the bonds between adjacent nucleotides in a DNA chain
Base pairing
-A-T (2 hydrogen bonds)
-G-C (3 hydrogen bonds)
Describe the hydrogen bonds between complementary bases
Base pairing
-A-T (2 hydrogen bonds)
-G-C (3 hydrogen bonds)
Name the complementary base pairs and compare the two types of pairs with respect to their relative strength and number of H bonds
Base pairing
-A-T (2 hydrogen bonds)
-G-C (3 hydrogen bonds)
Describe the polarity of DNA strands relative to one another in double stranded DNA
Antiparallel
Describe how DNA strand polarity (5’, 3’) gets its name
from the numbering of the carbon atoms in the deoxyribose sugar molecule that makes up the backbone of the strand
Describe the difference between RNA and DNA in terms of the 2’ position and be able to identify this in a diagram
RNA: has an OH on 2’ position
DNA: Has an H on 2’ position (more stable)
Briefly describe supercoiling and its relationship to DNA packing
Supercoiling: DNA helix twists upon itself to fit inside the cell
Describe the difference between positive and negative supercoiling with respect to overwinding or underwinding of the DNA helix
Positive: overwound
Negative: underwound (favored by bacteria for ease)
Identify the electrostatic charge of DNA
Negative because of all the phosphate bonds (each phosphate is negative)
Explain why most bacteria keep their chromosomes in a state of negative supercoiling
Make it easier to unwind
Identify the enzymes that alter the supercoiling state of DNA
Topoisomerases
Briefly explain how these topoisomerase enzymes change the supercoiling state of DNA
temporarily breaking one or both strands of the DNA double helix, allowing a section of DNA to pass through the break, and then rejoining the broken strands
Define DNA replication
Semiconservative, has three steps: initiation, elongation, and termination
Explain what it means for DNA replication to be semiconservative
Each daughter cell gets on original and one new strand
Name the main steps in DNA replication
Initiation: starts at the origin (ori), helicase unwinds DNA
Elongation: DNA polymerase III adds nucleotides in the 5’ –> 3’ direction
Termination: occurs at ter sites, Tus protein halts replication
Identify the loci on DNA where replication begins and ends
Begins at the ori, ends at the ter site
Define the functions of: the initiator protein DnaA; helicase; DNA primase; DNA polymerase III; DNA polymerase I; DNA ligase; RNAse H
-DnaA: initiates replication by melting separate DNA strands
-Helicase: unzips DNA strands
-DNA primase: synthesizes RNA primers
-DNA Polymerase III: main replication enzyme
-DNA Polymerase I: replaces RNA primers with DNA
-DNA Ligase: seals Okazaki fragments (seals nicks on the backbone)
-RNAse H: Digest RNA primers so that they they can be filled with DNA
Explain why DNA primase is needed in replication
DNA polymerase 3 cannot begin making DNA molecule on its own –> it can only elongate existing molecule
Primase makes an RNA primer that can be elongated by DNA polymerase 3
Describe the polarity of DNA synthesis
DNA polymerase can only synthesize DNA in the 5’ to 3’ direction
Distinguish between leading and lagging strands with respect to their strand polarity and the way they are replicated
Leading strand: continuously synthesized (5’–>3’)
Lagging strand: made in short Okazaki fragments (3’–>5’)
Identify Okazaki fragments, know their approximate length, and describe how they are connected
-Short DNA segments that are created during DNA replication
-Connected by the DNA polymerase 1 and then the enzyme DNA ligase
-Part of lagging strand (1kb segment)
Describe how DNA replication is terminated
At the ter site, a protein called Tus binds (creates trap), which stops the helicase and halts replication so it doesn’t just continue around and around the chromosome
Describe how interlinked replication chromosomes (catenanes) are separated
Certain proteins cut and re-join the DNA to separate the interlinked chromosomes
Define a plasmid, distinguish plasmids from chromosomes and describe what plasmids are used for
Plasmids are short (typically 3-20 kb), circular extrachromosomal DNA molecules that autonomously replicate
Used in molecular biology and genetics to carry genes of interest
–> replicates separately from chromosome
Define the central dogma of molecular biology
DNA –> RNA –> Protein
Define transcription
-DNA –> RNA
-Accomplished by key Enzyme: RNA polymerase holoenzyme
–Include core polymerase and sigma factor
**Makes a copy of the encoded info
Define translation
RNA –> Protein
Taking information from mRNA and converting it into polypeptide (into a protein)
Define the functions and subunits of the RNA polymerase holoenzyme
–Core polymerase: catalyzes RNA synthesis
–Sigma factor: recognizes promoters (~10 and ~35 regions)
Subunits have alpha, beta, and sigma subunits
**It’s a multiimportant enzyme and the sigma factor has a special role to play
Define the function of the sigma factor and describe when it dissociates from the RNA polymerase
Sigma factor typically dissociates from RNA polymerase shortly after transcription initiation
Define a gene
A segment of DNA that encodes a protein (gets transcribed into RNA)
Define a promoter
a DNA sequence that is read by a sigma factor
Essentially says: start transcribing here
Describe how a sigma factor recognizes a promoter in a double stranded DNA helix
Sigma factor recognizes consensus sequences at the -10 and -35 positions (relative to the start of transcription, the +1 position)
Sigma factor is able to scan along the double helix and make chemical contact with the nitrogenous bases in major and minor grooves of the DNA (can identify sequences in a closed double helical structure)
Explain why bacterial cells use different sigma factors to direct the expression of different groups of genes
Many species have multiple sigma factors and each has its own matching consensus sequence
–each sigma factor recognizes a specific promoter sequence on DNA
Describe a consensus sequence
A sequence of DNA, RNA, or protein that represents aligned, related sequences
–> kind of like a promoter sequence
Describe the structure of RNA and distinguish it chemically from DNA, both in its ribose component and in its nitrogenous bases
RNA is built of ribonucleotides
1) Nitrogenous base (uracil, no T)
2) Ribose sugar: like DNA but with OH at 2’ position; DNA has H)
3) Phosphate group: phosphodiester bonds just as ib DNA
Explain why the 2’ -OH of ribose makes RNA much less stable than DNA
The 2’-OH can occasionally attach the adjacent phosphodiester bond, breaking the RNA chain (self-cleaving)
–this happens well in warm temp and alkaline conditions
–DNA has no oxygen at the 2’ position (can’t self-cleave)
Describe the 3 principal components of a ribonucleotide and distinguish it from a deoxyribonucleotide
RNA is built of ribonucleotides
1) Nitrogenous base (uracil, no T)
2) Ribose sugar: like DNA but with OH at 2’ position; DNA has H)
3) Phosphate group: phosphodiester bonds just as in DNA
Describe the polarity of RNA
5’ –> 3’
Describe the relationship between promoters and sigma factors
Sigma factor recognizes sequences of promoter
Name and describe the 3 main steps in transcription
1) Initiation: RNA polymerase binds to promoter, opens DNA.
2) Elongation: RNA polymerase synthesizes RNA.
3) Termination: Ends via Rho-dependent or Rho-independent mechanisms.
Describe the steps in transcription initiation, distinguishing between the closed and open complex
1) RNAP “scans” for promoter sequences via sigma factor
2) Sigma factor binds to a promoter, forming the closed complex (DNA helix is “closed”)
3) RNAP unwinds DNA helix, making the open complex, and begins Synthesizing RNA. The sigma factor leaves once RNAP moves past the promoter
Identify the directionality of RNA synthesis
5’ to 3’
Explain how the complementary of bases allows for discrimination between correct and incorrect matches
Each base in DNA can only pair with its specific complementary base (lock and key mechanism) –> effectively filters out any mismatched bases that would disrupt the proper pairing and stability of the DNA molecule
Distinguish between the coding strand and template strand of DNA
1) Coding strand is the strand with the same sequence as the mRNA and runs 5’ to 3’
2) Template strand has the complementary sequence to the RNA and runs 3’ to 5’ –> used as the template to make mRNA used in coding strand
Define and distinguish between Rho-dependent and Rho-independent terminators with respect to the proteins involved and the DNA/RNA sequences involved
1) Rho-dependent terminators bind to a C rich sequence in mRNA and travel up RNA polymerase and cause it to fall off of the DNA and terminate transcription
2) Rho-independent terminators depend on a GC-rich region that forms a stem loop that is bound by a protein called NusA and causes RNAP to pause
Distinguish among the structures and functions of mRNA, rRNA, tRNA, and sRNA
mRNA: Carries genetic code.
rRNA: Component of ribosomes.
tRNA: Brings amino acids for translation.
sRNA: Regulates gene expression
Identify and define the major players in translation
-mRNA, containing the codons that specify the amino acid sequence
-Ribosomes, the rRNA-protein complexes that actually catalyze protein
formation
-Charged tRNA molecules that carry each of the 20 amino acids
-Elongation factors, proteins that help with translation
Describe how a tRNA molecule is able to decode a codon into the appropriate amino acid
-tRNA is a decoder molecule that converts the language of codons into an amino acid sequence (translates it)
-2 regions:
1) anticodon loop binds to its corresponding codon on mRNA
2) 3’ acceptor end is linked to the amino acid that corresponds to the anticodon loop
**When a tRNA has an amino acid attached to it, it’s called charged
Describe the function of an aminoacyl-tRNA synthetase, how it charges a tRNA, and how it ensures high fidelity
-Does the job of matching and attaching
-one for each of 20 amino acids
-Incredibly discriminatory, binding to their cognate tRNA molecule but to none of the others
Describe the organization of information in DNA and RNA into codons
Info organized into codons (3 nucleotide sequence in amino acids)
–> building blocks for protein synthesis
–> 4 nucleotides at each three positions
Define reading frame
The way that codons are read in an mRNA sequence
Define the genetic code, including its degenerate and universal
Composed of codons (3 nucleotide words that specify amino acids)
64 possible codons
–61 codons specify amino acids, other 3 stop codons
Universal: same in all species of life
Degenerate: each amino acid can be specified by multiple codons
Define and identify a start and stop codon
Start codon: AUG
Stop codon: UAG, UGA, or UAA
Describe the components of the ribosome in terms of their chemistry and their subunit division
Prokaryotic Ribosome (70S) Structure
- Small Subunit (30S) → 16S rRNA + 21 proteins (reads mRNA, aligns Shine-Dalgarno) –> first to bind
1 RNA molecule
- Large Subunit (50S) → 23S rRNA + 5S rRNA + 34 proteins (forms peptide bonds)
2 RNA molecules
- Composition: ~60% rRNA (structure + catalysis), ~40% proteins (stabilization).
- Function: Translates mRNA into proteins.
Describe how a ribosome becomes correctly positioned on mRNA and what components of the ribosome and mRNA interact to make this possible
Correctly positioned by an interaction between the 16S rRNA (part of the small subunit) and a sequence on the mRNA (RBS) –> happens before translation begins
Define a polysome and coupled transcription and translation and distinguish this from transcription and translation in eukaryotes
1) Polysome: cluster of ribosomes attached to a single mRNA –> translates genetic info on mRNA into multiple polypeptide chains during protein synthesis (beads on a string)
2) Couple translation/transcription: happens at the same time –> key feature in prokaryotes (bacteria)
**In eukaryotes, transcription happens first, then translation
Define transertion
Where newly synthesized polypeptides (proteins) can begin the membrane insertion process before they’re done (translation and insertion into the membrane)
Describe the 3 tRNA-binding sites on the ribosome and distinguish among them with respect to their functions
1) The A (acceptor) site binds to
incoming charged tRNAs
2) The P (peptidyl-tRNA) site holds the
tRNA with the growing polypeptide
chain
3) The E (exit) site holds the tRNA that
was just stripped of its polypeptide
before it leaves the ribosome
Describe the three principle steps in translation
1) Initiation: the ribosomal subunits come together at the RBS, aligning the
ribosome so that the initiator tRNA is positioned correctly in the P site.
2) Elongation: amino acids (on tRNAs) come into the A site (guided by a correct
codon-anticodon match) and are added to the growing polypeptide chain
3) Termination: releases the completed protein at a stop codon and then recycles
the ribosomal subunits to begin again
Describe peptide bond formation and identify a peptide bond
The nitrogen of the amine group on the tRNA in the A site attacks the carbonyl carbon of the peptide in the P site, transferring the peptide to the tRNA at the A site
A peptide bond is a chemical bond that links amino acids together to form proteins
Explain the function of EF-Tu in ensuring fidelity in translation
Charged tRNA are brought to the A site by EF-Tu GTP
Only leaves if there is a correct match, EF-TU hydrolyzes
–this all occurs in elongation stage
–essentially responsible for bringing the correct amino acid to the growing polypeptide chain during translation elongation
–chaperone
Describe the function of EF-G in translation
helps move messenger RNA (mRNA) and transfer RNA (tRNA)n through the ribosome during protein translation
–in elongation
Describe protein folding and the associated role of chaperones
Chaperones (e.g., GroEL-GroES) help proteins fold correctly based on finding their lowest energy conformation
–proper folding leads to it functioning
**essentially massage proteins to help
Describe the function of signal sequences and the SRP in the synthesis of membrane-bound proteins or the secretion of proteins destined to be exported from the cell
Provides a specific amino acid sequences with signal sequences that target proteins to the membrane –> these are recognized by SRP and brought into membrane
Define transformation
Uptake of DNA by an organism
Define horizontal gene transfer and distinguish it from vertical gene transfer
Horizontal gene transfer: gene gets transferred from one organism to another organism thats on the same level as that organism
Vertical gene: when DNA is passed from parents to offspring
Define competence and give reasons why a bacterial cell might want to take up free DNA from its environment
Competence: ability of an organism to take up free DNA from its environment and internalize it and possibly incorporate it into its own genome if necessary
–Many reasons why a cell wants to do it: diversity, food, etc
Define a transformasome and describe its function in cemptence
Transformasome: (gram positive organism) complex in a cell envelope that imports DNA –> allows cell to be transformed by importing a single strand of DNA from environment
Describe differences between gram + and gram - competence
Gram +: uses quorum sensing
Gram -: does not (competence machinery is different as well)
Define conjugation
Bacterial sex
Define the E.Coli F plasmid (F factor) and explain how it is transferred
F plasmid has an origin of transfer on it and it also encodes machinery that allows DNA to be transferred from donor cell to recipient cell (uses mating pilus)
Explain what happens when the F plasmid is integrated into the E.coli chromosome
Origin of transfer becomes part of the chromosome that allows segments of the chromosome to be transferred like F plasmid
Describe the unique feature of Agrobacterium tumefaciens DNA transfer
Transfer DNA to plants and transform plant cells –> used in agricultural technology
Describe phage transduction
Process where a virus (bacteriophage) transfer genetic material from one bacterium to another
Define bacteriophage
A virus whose host is bacteria
Distinguish between generalized transduction and specialized transduction
Generalized transduction: why which any gene may be transferred from donor to recipient
Specialized transduction: where only genes closely linked to the phage chromosomal insertion site may be transferred (similar to F plasmid excision)
Define restriction endonuclease
Proteins/enzymes that cleave DNA at specific sites on incoming “alien” DNA
Describe restriction sites in DNA and how they are used in molecular biology
Restriction sites are palindromic sequences –> used in molecular biology to cut DNA molecules in places where you want them to be cut (like scissors)
Describe CRISPR and its function in bacterial “immunity” to invading DNA
CRISPR: provides a rudimentary “immune system” by specifically attacking certain foreign DNA molecules (bacterium immunity)
–Kind of like a bouncer from a club
Briefly describe how CRISPR can be used in biotechnology
Used to target genes for modification or deletion in eukaryotic systems (can target anywhere because of sequences of gRNAS)
Define and describe homologous recombination
The integration of one DNA molecule into another –> combining two DNA molecules into one
Define the role of the RecA protein in recombination
Recognizes homologous regions and brings them close to one another so that strand invasion can occur and the two DNA molecules can attach to each other
Distinguish between generalized recombination and site specific recombination
Generalized recombination: which requires that the DNA molecules be recombined having substantial stretches of homology
Site specific: which requires that both DNA molecules have a short, specific sequence that is recognized by the recombinase enzyme
Briefly describe the importance of recombination in bioengineering
Used all the time to add or subtract genes from organisms
Define a mutation in DNA
Change to the nucleotide coding sequence
Distinguish among point mutations, insertions/deletions, inversions, and reversions
Point mutations: single nucleotide base changes (can sometimes have effect on proteins)
Insertion/deletions: add/delete from nucleotide sequence
Inversions: stretch of DNA sequence flips around
Reversions: another mutation (mutated sequence) goes back to its original sequence
Distinguish between transitions and transversions
Transition: point mutation where a purine is substituted for another purine or pyrimidine for another pyrimidine
Transversion: purine to pyrimidine (like A going to C or T)
Define and recognize silent mutations, missense mutations, nonsense mutations, and frameshift mutations
Silent mutation: point mutation that changes codon in coding sequence but does not change the amino acid (protein sequence is not changed at all)
Missense: changes codon and changes the specific amino acid (substituting one amino acid for another)
Nonsense: turns amino acid into a stop codon
Frame shift: insertions/deletions of nucleotides that change reading frame
Define a knockout mutation
Destroys the function of a gene
Describe how different chemical agents can cause DNA mutations
Intercalators - insert between bases in DNA helix —> cause misreading and introduces mutations
Describe how UV light damages DNA
Describe spontaneous reactions that cause mutations (e.g., cytosine deamination)
Cytosine deamination: That’s the loss of an amine group causing the nucleotides to mispair
Describe different mechanisms for DNA repair: photoreactivation,
nucleotide excision, base excision, methyl mismatch, recombination, and
translesion bypass synthesis
Photoreactivation: cleaves pyrimidine dimers –> undoes damage done by UV light
Nucleotide excision: removes damage (removes damaged nucleotides from DNA strand)
Base excision: removes damaged bases from nucleotide (replaces with new ones)
Methyl mismatch: repair fixes replication error (fixes mistakes from DNA polymerase)
Recombination: uses other copy of a gene to repair the damaged gene (if given two copies of gene)
Trenslesion bypass synthesis: error prone method –> if DNA is extensively damaged, it will allow DNA synthesis to proceed where normal DNA synthesis can’t
Distinguish between error-proof and error-prone repair pathways
Error proof: the pressed way, as it restores the original sequence
Error prone: the last resort way –> preferable only to death, as errors may be introduced by the repair itself
Describe the mechanism of methyl mismatch repair
Based on the fact that the oldest gene in the cell (parent gene) is methylated. If, in replication, the original strand and new strand don’t match, it will use the methyl gene as the standard and will correct the newly synthesized strand so that it will match the methylated strand. Thus becoming methylated as well.
Define a mutator strain
Strain that is lacking one or more DNA repair pathways
Describe the SOS response and its relationship to error-prone DNA repair
SOS response is last resort (happens when there is extensive DNA damage and cell is at risk of losing integrity of its chromosome) to save its chromosome. It is error prone.
Describe the logic of having an error-prone DNA repair system if it can introduce mutations
It’s mutate or die. Cell would rather mutate.
Briefly describe non-homologous end joining
Broken DNA helices are put back together without any consideration to their sequences –> could introduce mutations (last ditch effort to save cell)
Define transposable element and name two examples
Transposable element is a DNA molecule that is able to jump out of one strand of DNA and insert itself into another DNA molecule
Two ex: IS, transposon
Describe an inverted repeat
Typically on either side of transposons or insertion sequences –> sequences of DNA that match one another (repeated sequences and inverted)
Describe the utility of transposons as a way to screen for genetic function
You can randomly inactivate genes when a transposon happens to hop into a gene –> used to screen mutagens
Describe how GC content can be a way of identifying horizontal gene transfer events
Can tell about gene origins –> places with more/fewer GC base shows it may have originated from another molecule
