Exam II Study Questions

What is the difference between a nucleoside and a nucleotide?

nucleoside → pentose sugar & nitrogenous base

nucleotide → pentose sugar, nitrogenous base, & a phosphate

Why are carbon atoms on the sugar labelled prime?

to distinguish them from the carbon atoms on nitrogenous base;

5’ → outside of ring and attaches to a phosphate group
3’ → attached to an OH group involved in making phosphodiester bonds

What bases are in DNA and what bases are in RNA?

DNA → adenine, cytosine, guanine, & thymine

RNA → adenine, cytosine, guanine, & uracil

Which bases pair with each other?

adenine + thymine/uracil

cytosine + guanine

How many hydrogen bonds are there in each base pair?

adenine + thymine/uracil → 2 hydrogen bonds

cytosine + guanine → 3 hydrogen bonds

Which bases are purines and which bases are pyrimidines?

purines → adenine & guanine

pyrimidines → cytosine & thymine/uracil

How many bases are in one turn of the DNA double helix?

10 base pairs

Why is the major groove of DNA important?

it allows proteins to recognize and bind to specific DNA sequences

What do we mean when we say that the two DNA strands in a double helix are antiparallel?

the two DNA strands run in opposite directions

• one strand runs 5′ → 3′.

• the complementary strand runs 3′ → 5′.

What does the enzyme gyrase do to DNA?

• relieves twisting and tension in DNA during replication and transcription

• relieves positive supercoiling ahead of the replication fork

• introduces negative supercoils into DNA

In what direction does gyrase twist the DNA?

in the negative direction

Does gyrase require ATP (energy)?

yes

What is a gene?

functional unit of genetic information

What is a genome?

total complement of genetic elements in the cell

What is the difference between a chromosome and a plasmid?

chromosome → main genetic element in prokaryotic cells containing all the genes necessary for life

plasmid → circular or linear double stranded DNA molecules that replicate separately from the chromosome and provide “extra” characteristics for the cell

How do we tell if a genetic element is a chromosome or a plasmid?

chromosome → main DNA containing all genes necessary for life

plasmid → DNA molecule that replicates independently from the chromosome and provides extra characteristics

What is an operon?

a group of genes transcribed together to form a single RNA or single mRNA

What is meant by semiconservative replication?

each newly formed DNA molecule contains one parental strand and one newly synthesized strand in the double helix

What is the difference between the enzymatic functions of DNA polymerase I and III?

DNA polymerase I → removes RNA primers and replaces them with DNA

DNA polymerase III → synthesizes new DNA strands

What are the important characteristics of DNA polymerases? (Most importantly which direction do ​they synthesize the new DNA?)

• require a primer (DNA or RNA)

• require nucleotide triphosphates as the building blocks
  dNTPs: dATP, dCTP, dGTP, dTTP

• synthesize DNA in the 5’ to 3’ direction (template read 3’ to 5’)

• catalyze nucleophilic attack of the 3’ OH to the 5’ phosphate releasing pyrophosphate (2 phosphates joined together)

• proofread and have 3’ to 5’ exonuclease activity to remove an erroneously added mismatched base (the enzyme “backs up”)

Why is proofreading important for DNA polymerases?

it prevents mutations and maintains the accuracy of DNA replication

What are the functions of helicase, SSB, primase, and ligase in DNA replication?

helicase → unwinds the DNA at the replication fork by breaking hydrogen bonds between complementary bases

SSB → keeps the two single strands from winding up again by binding to separated DNA strands

primase → synthesizes short RNA primers and provides a free 3’ OH for DNA polymerase

ligase → seals gaps between discontinuous DNA fragments by forming phosphodiester bonds

What is an Okazaki fragment in DNA replication?

short segment of newly synthesized DNA formed discontinuously on the lagging strand during DNA replication

What is the difference between the lagging and leading strands in DNA replication?

leading strand → synthesized continuously in the 5’ to 3’ direction toward the replication fork

lagging strand → synthesized discontinuously in the 5’ to 3’ direction away from the replication fork

What enzyme removes the RNA primer during replication?

DNA polymerase I

What is oriC in E. coli?

origin of chromosome replication;

location on the E. coli chromosome where DNA replication begins

Is E. coli chromosome replication unidirectional or bidirectional?

bidirectional

What is the difference (what are the functions) of mRNA, tRNA, and rRNA?

mRNA → linear, short-lived, template/instructions for translation to make proteins

tRNA → stable, long-lived, folded up to prevent degradation and delivers amino acids to the ribosome during translation

rRNA → stable, long-lived, folded up to prevent degradation and plays a structural and functional role in ribosomes

What is the difference between bacterial RNA polymerase holoenzyme and core enzyme?

holoenzyme → α₂, β, β′, ω, σ; does transcription initiation; recognizes promoters

core enzyme → α₂, β, β′, ω; does RNA elongation

Why is sigma subunit important for RNA polymerase?

it helps bacterial RNA polymerase recognize and bind to the correct promoter region on DNA to initiate transcription;

• Sigma 70 (σ⁷⁰) binds to Pribnow Box TATAAT sequence at -10 and -35 region TTGACA sequence

What determines how often transcription will start at a promoter?

how well RNA polymerase binds; strength of the promoter

What direction is the DNA template read by RNA polymerase?

3’ to 5’

In what direction is the new RNA synthesized by RNA polymerase from a DNA template?

5’ to 3’

What does rho protein do in transcription?

involved in terminating transcription in bacteria

• binds to newly formed RNA

• moves down the RNA toward the RNA polymerase

• catches up to RNA polymerase when it pauses

• pushes RNA polymerase off the DNA by unwinding the RNA

• helps separate the RNA transcript from the DNA template, ending transcription

How can the structure of the mRNA produced by RNA polymerase cause transcription termination?

• the newly synthesized RNA can fold back on itself to form a stem-loop structure with very strong GC base pairs after they are formed

• causes RNA polymerase to pause and helps terminate transcription

• if the stem-loop is followed by uridine bases in the RNA then this destabilizes the RNA polymerase-DNA-RNA complex because AU base pairs are extremely weak

What types of transcripts are processed in bacteria and why?

tRNA → become functional amino acid carriers

mRNA → become functional ribosome components

What are the characteristics of a polycistronic mRNA?

transcript from more than one gene that are arranged in an operon

• mRNA contains multiple Open Reading Frames (orfs) that encode proteins

Do all protein coding genes in a mRNA require a ribosome binding site for translation?

yes, each protein coding gene must have a ribosome binding site (RBS) (Shine-Dalgarno site) in the mRNA to signal the ribosome to start translation at the correct AUG codon

What is the difference between transcription factors (in archaea and eukaryotes) and sigma subunit ​(in bacteria)?

transcription factors → help recruit and position RNA polymerase at promoters; separate proteins that help guide and regulate transcription

sigma subunit → help RNA polymerase recognize promoters; part of bacterial RNA polymerase

What is a peptide bond?

covalent bond that connects the carboxyl group of one amino acid and amino group of another amino acid together to form a protein

What are the two major protein substructures?

alpha helix and beta sheet

How does the mRNA codon match up to the tRNA anticodon?

through complementary base pairing during translation

Which end of the tRNA has the amino acid attached? (5' or 3'?)

3’ end with a CCA sequence

Is the amino acid attached to the tRNA by the C-terminal or N-terminal end?

C-terminal end

What does an aminoacyl tRNA synthetase do?

recognizes different parts of the tRNA and adds the correct amino acid to the acceptor stem

How does an aminoacyl tRNA synthetase "know" which amino acid to attach to which tRNA?

it recognizes specific structural features of both the amino acid and its corresponding tRNA to ensure the correct amino acid is attached

How many aminoacyl tRNA synthetases are there in a cell?

20

What is meant by the statement that the genetic code is "degenerate?"

several amino acids can be encoded by more than one codon

What is the wobble hypothesis?

the pairing between the 3’ base in the codon and the 5’ base in the anticodon can “wobble” or form a mismatched base pair

Which base in the codon wobbles?

the 3rd base

What is a reading frame?

the way a sequence of mRNA nucleotides is divided into groups of three bases (codons) during translation

What is an open reading frame (orf)?

a start codon followed by a number of amino acid encoding codons ending with a stop codon

How many potential reading frames are in a mRNA sequence?

3

How many potential reading frames are in a DNA sequence?

6

What are the sizes of the two bacterial ribosome subunits?

30S and 50S

How does a ribosome "know" where to start translation on a mRNA?

the 30S small subunit binds mRNA with the 16S rRNA base pairing with the ribosome binding site RBS (Shine-Dalgarno)

What is the function of the 16S rRNA in the ribosome?

• makes sure that the ribosome will be situated in the correct frame

• makes sure that the “start” codon is recognized

What amino acid is always the first one in bacterial translation?

fMET (N-formylMethionine)

What are the E, P, and A sites in a ribosome?

E → exit

P → peptidyl

A → aminoacyl (acceptor)

How do the E, P, and A sites work during addition of a new amino acid to the growing polypeptide?

A → new tRNA carrying an amino acid enters

P → holds the tRNA carrying the growing polypeptide chain

E → empty tRNA leaves the ribosome

What is the function of the 23S rRNA in the ribosome?

to act as a ribozyme (catalytic RNA) that catalyzes peptide bond formation between amino acids during translation

What energy molecule is utilized in translation?

GTP

Does transcription and translation occur at the same time in bacteria?

yes

What is the difference between negative control and positive control of transcription?

negative → repressor protein binds DNA and blocks RNA polymerase → blocks transcription

positive → activator protein binds DNA and helps RNA polymerase bind → helps transcription

What is the difference between repression and induction of transcription?

repression → repressor prevents transcription → turns transcription off

induction → inducer causes transcription to increase → turns transcription on

What is an operator?

a specific DNA sequence in an operon where a repressor protein binds to regulate transcription

What is a promoter and how does it relate positionally on the DNA to the operator?

a specific DNA sequence where RNA polymerase binds to begin transcription;

located upstream of the structural genes, next to the operator

Why do transcription regulatory proteins often bind to inverted repeat sequences in the DNA?

because many regulatory proteins function as dimers (two protein subunits joined together)

A common signature of bacterial regulatory proteins is the helix-turn-helix structure. How does this protein interact with DNA?

recognition helix → interacts with specific sequences in DNA

stabilization helix → stabilizes the recognition helix through hydrogen bonds

What is catabolite repression?

a mechanism of global control that controls the use of carbon sources if more than one is present;

ensures that the best carbon source is used first

What is the signal molecule for catabolite repression?

cAMP

What enzyme makes the signal molecule?

adenylate cyclase makes cAMP when glucose levels are low

Is catabolite repression activation or repression? Is catabolite repression positive or negative ​control?

repression & positive control

For the lac operon, explain why there are two layers of transcription control.

so that bacteria can efficiently use glucose first and only produce lactose-metabolizing enzymes when needed

negative control → is lactose present?

• no → lac repressor binds the operator and blocks transcription

• yes → allolactose binds the repressor, causing it to release the operator

positive control → is glucose present?

• high → cAMP levels low, CAP can’t effectively bind DNA

• low → cAMP levels high and bind CAP

What are the two parts for a two component regulation system?

1. sensor kinase

2. response regulator

What does each protein do in a two component regulatory system?

sensor kinase → senses an environmental signal and adds a phosphate group to itself (autophosphorylation)

response regulator → receives the phosphate group and changes gene expression, often by binding DNA and regulating transcription

What role does phosphate play in two component regulatory systems?

a molecular switch that transfers the signal from the sensor kinase to the response regulator

What is quorum sensing? Why is it important for the bacterium?

a regulatory mechanism that assesses population density;

a way that bacteria “talk” to each other within a species or cross-species;

bacteria need a certain amount of each other to perform a function

What is the function of the autoinducer in quorum sensing?

to diffuse in and out of the cell and act as a signal molecule;

when concentration of autoinducer is high, then regulatory proteins turn on transcription of the target genes

How can small RNAs (sRNA) inhibit translation?

by binding to mRNA in an antisense manner at the RBS

How can small RNAs (sRNA) control degradation of a mRNA?

stimulate degradation → binding to mRNA in an antisense manner to stimulate degradation

inhibit degradation → binding to mRNA in an antisense manner and preventing the binding of a ribonuclease at a sequence in the mRNA

Define a riboswitch.

RNA molecules that bind small metabolites and regulate gene expression (specifically regulate translation)

How does attenuation control transcription?

mechanism of transcription regulation in bacteria that controls whether transcription continues or stops early by forming different RNA secondary structures (hairpins)

• tryptophan HIGH → 3—4 terminator hairpin → transcription OFF

• tryptophan LOW → 2—3 antiterminator hairpin → transcription ON

Why is translation speed an important part of attenuation?

how quickly the ribosome moves determines which RNA hairpin forms, controlling whether transcription continues or stops

• FAST → terminator forms → transcription OFF

• SLOW → antiterminator forms → transcription ON

What is feedback inhibition of an enzyme or metabolic pathway?

when a downstream metabolite interacts with an upstream enzyme to (temporarily) modulate its activity

What is the difference between genotype and phenotype?

genotype → genetic makeup of an organism

phenotype → observable characteristics of an organism

What is the difference between a mutation and a mutant?

mutation → a heritable change in the base sequence of nucleic acid of the genome of an organism

mutant → a strain carrying a mutation

What is the difference between an auxotroph and a prototroph?

auxotroph → a mutant that has an additional nutritional requirement for growth

prototroph → parental strain (usually wild type)

What is the difference between a spontaneous mutation and an induced mutation?

spontaneous mutation → occur without external intervention, mostly from occasional replication errors by DNA polymerase

induced mutation → made environmentally and deliberately, result from exposure to natural radiation or DNA-modifying chemicals

Why are two rounds of replication needed to make a spontaneous mutation arising from a tautomerization permanent in the DNA?

a tautomeric shift initially causes an incorrect base pairing, but the mutation must be copied into a new DNA strand to become permanent

• 1st replication → creates a mismatched base pair

• 2nd replication → fixes the altered sequence into a normal base pair, making the mutation permanent

How does a base analogue make a mutation?

resembles a normal DNA nitrogenous base but can pair with the wrong nucleotide, causing a mutation during DNA replication

What is the difference between a silent mutation, a missense mutation, and a nonsense mutation in a protein coding gene?

silent mutation → don’t affect the sequence of the encoded polypeptide

missense mutation → sequence of amino acids in the ensuing polypeptide is changed, often resulting in an inactive protein or one with reduced acitvity

nonsense mutation → results in a premature stop codon and typically results in a truncated (incomplete) protein that lacks normal activity

How do insertion or deletion mutations effect translation?

they result in a shift in the reading frame and scramble the polypeptide sequence downstream

What is homologous recombination?

a process that results in genetic exchange between similar DNA from two different sources

What protein is the most important for the homologous recombination process?

RecA protein

What is a heteroduplex in recombination?

a double-stranded DNA molecule formed during homologous recombination that contains one strand from each of two different DNA molecules

What two different types of molecules can result from recombination?

patch → non-crossover; DNA is repaired or exchanged without swapping the surrounding DNA regions

splice → crossover; DNA molecules exchange corresponding DNA segments, resulting in recombinant molecules