BiOL 4461- Exam 1
The transcriptome is defined as:
all RNA molecules, including rRNA, tRNA, mRNA, and ncRNA in one or population of cells
Restriction Enzymes
are naturally occurring endonucleases (or molecular scissors) that cleave DNA into fragments at specific sites DNA methylation protects the bacteria's own DNA from cleavage
Where is DNA unwound
at the replication fork
Circle the assay that can not be used to demonstrate whether a protein can bind nucleic acid? a) Electro-mobility shift assay b) DNA footprinting c) In-vitro transcription assay d) Fluorescence anisotropy e) all of the choices are correct
c) In-vitro transcription assay
Translation
conversion of mRNA sequence into the amino acid sequence of a polypeptide (protein synthesis)
Which of the following is not necessary to ensure translation occurs correctly? a. tRNA anticodon must match the amino acid bound to it b. tRNA anticodon must match the codon on the mRNA c. activated tRNA must enter the A-site on the ribosome d. tRNA must enter the A-site of the ribosome prior to being activated
d. tRNA must enter the A-site of the ribosome prior to being activated
A stretch of chromosome that codes for a trait can be called a(n):
gene
Which enzyme breaks hydrogen bonds and unwinds DNA
helicase
Which enzyme joins to DNA fragments togther
ligase
Which of the molecules transfers the genetic code to the cytosol in eukarytoes
mRNA
mRNA
messenger RNA carry sequence information to the translation complex
ncRNA
noncoding RNA not translated into a protein, can directly preform functions
OriC
origin of replication
DNA Replication (basic)
polymerizing nucleotides of DNA into long chains (strands) using the sequence on the other strand as a guide 2 strands are separate, each serving as a template for synthesis SEMICONSERVATIVE (one old and one new strand)
Replicon
portion of the genome that contains an origin and is replicated as a unit
Which enzyme synthesizes of short RNA sequence that is complementary to the DNA
primase
PCR
process that amplifies small pieces of DNA to create multiple copies of that DNA sequence Components: -DNA template -DNA primers -DNA polymerase I -dNTPS -Mg2+
The ribosome is comprised of:
rRNA and proteins
DnaA
replication initiation factor for DNA
rRNA
ribosomal RNA; type of RNA that makes up part of the ribosome (80% of RNA in cells)
SSBs
single stranded binding proteins Coat single stranded DNA to protect it from damage
What event must occur for translation to initiate?
small ribosomal subunit mut bind to mRNA with fmet-tRNA in P site.
Primase
synthesizes short complementary strands of RNA primers (approximately 10 nucleotides) needed by DNA polymerase
How are fluorescent dyes commonly used in DNA sequencing?
to label DNA fragments for detection
Which enzyme can relax or supercoil DNA
topoisomerase
tRNA
transfer RNA carry activated amino acids to ribosome for polypeptide synthesis
Catenanes
two intertwined DNA molecules Happens once replisome halts
List the components of a bacterial holoenzyme RNA polymerase and the number of subunits used per holoenzyme?
1 β, 1 β', 2 α, 1 sigma factor
Events in order for qRT-PCR
1. Extract nucleic acids 2. Digest DNA 3. Use PCR to convert RNA to cDNA 4. Amplification and detection by fluorescence
Place events in order for a microarray
1. Print DNA probes on solid array 2. Fluorescently label cDNA or RNA and hybridize 3. Array is imaged 4. Probes are quantified
What does DNA polymerase require
1. template 2. primer 3. dNTPs
Replisome
12 proteins involved in replication 2 replisomes move in either direction away from the origin
What direction is DNA synthesis
5' to 3'
DNA polymerase III
5' to 3' Lagging strand is synthesized in short fragments (Okazaki fragments)
DNA polymerase I uses ______ exonuclease activity to displace and remove _______ sequences while filling in the gaps between DNA Okazaki fragments, while DNA polymerase III uses _______ exonuclease activity as a proofreading mechanism to remove mismatched nt inserted into the growing nucleotide chain.
5' to 3' RNA 3' to 5'
DNA Ligase
A linking enzyme essential for DNA replication; catalyzes the covalent bonding of the 3' end of a new DNA fragment to the 5' end of a growing chain.
A gene is
A segment of DNA that codes for a protein
Which terms/abbreviations (circle all that apply) are used to describe a DNA binding site used by the basal eukaryotic transcription machinery? a) DPE b) GRE c) TATA box d) Inr e) all of the choices are correct
A, C, D
Chargaff's rules
A=T and C=G Measured concentrations of each of the four nucelotides Most-->least: A, T, G, C
RNA
Acts as a messenger, taking information from DNA to then synthesize proteins -tRNA -rRNA -mRNA (code for protein) -ncRNA
Who Demonstrated DNA was the hereditary molecule by radiolabeling phosphorus on DNA and sulfur on bacteriophage protein
Alfred Hershy and Martha Chase
Define genome
All the genetic info in an organism or cell (including viruses)
What molecule is needed for translation to work?
Amino-acylated tRNA
Who Demonstrated that the transforming principle could only be blocked by DNase treatment
Avery, McLeod, McCarty
DNA Polymersae I
Both directions Removes RNA primers, fills gaps with DNA Okazaki fragments are joined by DNA ligase
Which of the following is not true of the significance of the sigma factor: a. Sigma factor helps position the core RNAP at the promoter in prokaryotes. b. Sigma factor influences specificity and stability of the RNA polymerase in prokaryotes. c. When associated with the core RNAP, the sigma factor enhances gene transcription by binding to UP elements in prokaryotes. d. Sigma factor must dissociate from the core RNAP for RNAP to clear the promoter and enter elongation phase of transcription. e. Prokaryotes use different sigma factors in response to environmental stimuli.
C. When associated with the core RNAP, the sigma factor enhances gene transcription by binding to UP elements in prokaryotes.
cellular vs acellular
Cellular -Fungi, protists, bacteria, archaea (pro + euks) Acellular -Viruses, viroids, satellites, prions
Sanger Sequencing
Chain termination method Uses ddNTPs that terminate chain polymerization (missing OH which stops synthesis) Disadvantages: short reads, radioactive signal to detect
Why can't both strands of DNA be replicated continuously?
Chromosome structure is a DNA double helix, in which the two DNA strands are antiparallel. So 5' to 3' DNA synthesis by DNA polymerase can take place continuously on only one of the strands at the replication fork (the leading strand). On the lagging strand, short DNA fragments must be made by a "backstitching" process resulting in Okazaki fragments.
Hershey and Chase Experiment
Concluded that DNA carries the genetic materials of the cell Phage experiments tracking the movement of DNA during phage infection
RNA Polymerase parts
Core enzyme- catalyzes RNA synthesis Sigma factor- helps core recognize the start of genes Holoenzyme = core + sigma 1 beta, 1 beta', 2 alpha, 1 sigma = holoenzyme
DNA Methylation
Covalent modification of DNA that does not change the DNA sequence -Controls initiation -Produces heritable mark Addition of methyl group to base in DNA by DNA methyltransferases
The element that transformed the bacteria in Griffith's experiments was:
DNA
What is the central dogma
DNA --> RNA --> Protein DNA (Replication, DNA polymerase) RNA (Transcription, RNA polymerase) Protein (translation, ribosome)
Which enzyme catalyze the transfer of methyl group onto DNA
DNA methyltransferase
Which enzyme adds complementary bases to the growing nucleotide strand
DNA polymerase
What is proofreading carried out by in replication
DNA polymerase III
DUE
DNA unwinding element
Frederick Sanger and colleagues used which of the following to terminate DNA chain polymerization?
Dideoxynucleotides
Describe Meischer's discovery
Discovered nucleic acid Worked with WBCs and isolated nuceli, saw nucleic acid
Describe Hammerling's experiments
Discovered where the DNA was located (Removed the cap structure from green algae and it grew back. Removed the foot and it would not grow back.) Showed that the foot contained the nucleus, and the nucleus contained the DNA
Who Concluded that nucleotides must be arranged in a way that A=T and G=C
Erwin Chargaff
Fluorescent Sanger Sequencing
Fluorescently label the ddNTPs so that they each appear a different color and can be read by a laser at a fixed point
Who said Some "principle" could transform a non-virulent microbe into a virulent microbe
Frederick Griffith
Who Used garden peas to discover the principle of inheritance - units (genes) are passed to offspring
Gregor Mendel
What type of bond forms between complementary base pairs in the genome?
Hydrogen
Avery, McCarty, McLeod experiments
Identified that the transforming principle is stored as DNA
Watson, Crick, Wilkins determinations using Fraklin and Chargaff.
Learned complementary base pairing in DNA (A=G, T=C) Purine = Pyradine Proposed that DNA was right handed helix and DNA RUN ANTIPARALLEL TO EACH OTHER (5'-3')
Which method revolutionized DNA sequencing by allowing simultaneous sequencing of multiple DNA fragments?
Next generation sequencing
Unlike in prokaryotes, transcription takes place in the _________ and translation takes place in the __________ for eukaryotes. The RNA message must also be processed to produce mature mRNA. A _________ is added to increase mRNA stability and recognition by ribosome later. The _________ end of the mRNA is _________ which also aids in mRNA stability. Lastly, _________ are removed and _________ are spliced together by _________.
Nucleus Cytosol 5' cap 3' Polyadenylated Introns Exons Spliceosomes
What different roles do DNA polymerase I and polymerase III have in DNA replication?
Pol I -Inserts dNTPs in the 5' to 3' direction (DNA repair) -Fills gaps between Okazaki fragments -Exonuclease activity in both directions Pol III -Inserts dNTPs in the 5' to 3' direction (DNA replication) -High fidelity enzyme -Requires RNA primer for DNA synthesis -Exonuclease activity only in 3' to 5'
Describe the chemical structure of DNA
Polymer made up of nucleotides -Pentose sugar (lacks OH group) -PO4 group -Organic (N) base
Which of the following techniques dramatically increases amount of DNA by orders of magnitude; 30 minutes provides enough DNA to do nucleotide sequencing, restriction analysis, cloning, etc?
Polymerase Chain Reaction
Next generation sequencing (NGS)
Process millions of reactions in parallel = high speed and throughput Used to determine order of nucleotides in entire genomes Process: -Library preparation -Amplification -Sequencing -Reassembly
Prokaryotes vs Eukaryotes transcription and translation
Prokaryotes -Coupled tightly -Occurs in cytoplasm Eukaryotes -Spatially separated -Transcription in nucleus -Translation in cytoplasm
Prokaryotes vs Eukaryotes: Genome size variation, organization, and structure
Prokaryotes: -No true nucleus -No introns or histones -Circular dsDNA -supercoiled Eukaryotes -Complex -True nucleus -Introns or histones -Linear chromosomes -Nucleosome
Prokaryotic cell vs Eukaryotic cell
Prokaryotic -Open floor plan with simple morphology -Lack a true membrane-enclosed nuclease -Nucleoid Eukaryotic -True membrane-enclosed nucleus -Larger and more complex
How do we know that DNA is the genetic material?
Proteins require RNA and other proteins to be made DNA cannot do cellular work (just storage) RNA requires DNA as a template and protein as a catalyst
Topoisomerase
Regulates supercoiling without generating permanent strand breaks
Topoisomerases
Relives twist generated by rapid unwinding of helicase, prevents supercoiling Breaks and reseals strands without altering nucleotide sequence Resolves concatimers
Which of the following enzymes copy RNA into complementary DNA (cDNA)?
Reverse transcriptase
Who DNA molecule image by X-ray diffraction showing double helical structure
Rosalind Franklin
Which of the following techniques does not measure gene transcript levels?
Sanger sequencing
Rho-dependent termination : transcription as is _________ : translation. -10/-35 region : RNAP as is _________ : ribosome. Sanger sequencing : genome as _________ : transcriptome.
Stop codon Shine-delgarno RNA-seq
DNA
Stores hereditary information that is replicated and passed on to next generation
Summarize Mendel's principle of inheritance
Studied garden peas, saw that there were genes that determined heredity
Transcription
Synthesis of ssRNA molecule using DNA template
How is an RNA strand synthesized?
TRANSCRIPTION 1. Regulated by regulatory elements 2. DNA unwinds next to a gene 3. RNA is transcribed 5' -3' from DNA template strand (RNA polymerase reads in 3' to 5')
RNAP transcribes __________ DNA strand in the ____________ direction to produce mRNA sequence that is similar to the _______ DNA strand.
Template 3' to 5' Coding
In eukaryotes, RNAP II cannot identify promoter on its own and requires a number of accessory proteins called:
Transcription factors
Describe Griffith's experiements
Transforming principle Tried to develop pneumonia vaccine, wondered if one strain could transform into another. Concluded that some principle changed the non-virulent organisms into virulent ones
T/F: Rho has helicase activity and can unwind DNA:RNA hybrid complex. Rho has ATPase activity that helps it move across the nascent mRNA. Rho recognizes the rut sequence that is present on the DNA. Ribosomes can inhibit transcription termination by Rho.
True True False True
Helicase
Unwinds DNA and provides force to move replisome (Disrupts H-bonds)
Which of the following is found on RNA but not DNA?
Uracile
Oxford's Nanopore Technology (NNGS)
Uses a single DNA molecule as a sequencing template.
Who were the two men who established the structure of DNA?
Watson and Crick
When does replication stop
When replisome reaches termination site (ter) on DNA Tus protein binds ter site --> halts
If DNA were replicated conservatively, how would the results of the Meselson & Stahl experiment have differed from the actual results they obtained
Would expect that after 1 round of replication, 1/2 of all new DNA would be composed of old strands, and 1/2 would be new (No mix of old + new)
Rosalind Franklin
X-ray diffraction to take a picture of the DNA molecule. Showed double helix structure
