Biochem 153B Final e
Quiz yourself by thinking what should be in
each of the black spaces below before clicking
on it to display the answer.
Help!
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| show | purines: N1, N3
pyrimidines: N1, N3, N7, but not N9 because the electrons are delocalized due to aromaticity -- protonation would lead to loss of stability
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| show | aromatic bases are planar and can stack efficiently
p-orbital delocalization leads to adjacent stacked bases strongly attracting with induced dipole interactions (leads to 3D structures)
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| What are the relative stability of the tautomers? | show 🗑
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| show | If enol form pre-dominated, could get not-watson crick base pairs
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| free nucleotide tautomeric stability | show 🗑
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| show | glycosidic bonds were symmetrical about pseudodyad axis which allows for any order of base pairs without disrupting helix
AT and GC are same width
distance between C1' atoms is same for all base pairs
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| significance of Chargaff’s rules in formulating watson crick base pairing model | show 🗑
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| show | the Z glycosidic bonds are related by a pseudodyad axis and the distance between C1' atoms is the same, so AT and GC are same widths and won't disrupt the helix
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| How do the overall shapes of A- and B-form helices differ and how do the different sugar puckers contribute to this? | show 🗑
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| show | B DNA is stabilzied by interaction with H2O and H2O binds more tightly to B DNA ==> dehydrating conditions lead B --> A
has 2 layers of H2O in the major groove -- bridges adjacent base pairs on opposite sides and bridges layer 1 of water molecules
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| What is the role of the 2’OH group in stabilizing A- relative to B-RNA? | show 🗑
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| show |
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| show | lessens interphosphate electrostatic repulsions
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| show | purines: C3' endo, syn
pyrimidines: C2; endo, anti
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| B structure | show 🗑
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| A structure | show 🗑
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| What are the physical parameters that favor single-stranded DNA? | show 🗑
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| Conformational entropy - what is it and what suggests that it is the main entropic contribution to basepair formation | show 🗑
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| Translational entropy - what is it and why does it increase when DNA melts (ssDNA formation) | show 🗑
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| how does Electrostatic repulsion between the strands affect DNA melting | show 🗑
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| What are the physical parameters that favor double-stranded DNA | show 🗑
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| Hydrogen bonding between the strands - why is this a minor factor in aqueous solution for formation of dsDNA | show 🗑
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| show | induced dipole interactions between base pairs and it's enthalpically favorable for dsDNA
also hydrophobic interactions which drives dsDNA entropically --> lipid aggregation releases water from ordered stat
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| show | absorbance of DNA solution increases during DNA denaturation as a result of disruption of electronic interactions between adjacent bases
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| show | sigmoidal-shape indicates cooperativity: each step increases the favorability of the next step
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| show | 1/2 max absorbance means equal [dsDNA] and [ssDNA]
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| how was marmur doty equation determined | show 🗑
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| show | with long and complicated duplexes (various base pairs)
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| under what circumstances does marmur doty break down? | show 🗑
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| What is the physical meaning of the values in the nearest neighbor table? | show 🗑
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| How do we use the values in the nearest neighbor table to calculate the free energy, enthalpy, and entropy of helix formation and to determine melting temperature | show 🗑
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| Why does the length dependence of melting temperature vanish for sufficiently large DNA? | show 🗑
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| show | PCR to amplify DNA and look at number of repeat sequences for each individual (varies from human to human)
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| Why does DNA profiling depend upon the ability to calculate DNA melting temperatures accurately? | show 🗑
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| show | it influences the DNA structural transitions such as the formation of denatured bubbles (DNA melting) and nucleosome assembly
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| What is the physical meaning of Twist, T | show 🗑
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| What is the physical meaning of writhe, W | show 🗑
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| show | L = T + W
topologically invariant
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| How do interwound and toroidal supercoils contribute to the Writhe? | show 🗑
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| What do we mean when we say that Linking number is a topological invariant? | show 🗑
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| How do type I topoisomerases change linking number? | show 🗑
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| show | breaks both strands and passes double stranded segment through and reseals; each cycle changes L by +/-2
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| show | ALL proceed through covalent intermediates which involve ester linkages between phosphates at the break and tyrosines in the active site -- phosphotyrosine linkage preserves energy of phosphoester bond and hold broken end in place
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| show | lowest free energy topological state is W-->0
-- increased free energy as a result if increased writhe
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| How can supercoiling drive DNA melting? | show 🗑
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| How is nucleosome assembly influenced by DNA topology? | show 🗑
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| show | 1. e. coli genome is 1 huge circular chromosome and replication occurs semi-conservatively from a single origin on each chromosome
2. replication occurs bi-directionally
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| What three properties are common to all DNA polymerases? | show 🗑
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| show | DeltaG is slightly negative, with a high K value, therefore if [PPi] > 200, than dNTP would catalyze net pyrophosphates degradation --> maintain low PPi levels in-vivo
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| Given that DNA polymerases use 5’ NTPs as substrates, why does it make sense to always polymerize DNA in the 5’ to 3’ direction? | show 🗑
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| Why is it useful for the end of the primer/template duplex in the DNA polymerase active sites to assume an A conformation? | show 🗑
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| show | 2 metal ion mechanism -- metal ions are used to catalyze phosphoanhydride bond by creating a near-optimal transition state via lowered entropy and results in a suboptimal product to stabilize the 3'-OH to form new phosphoester group and translocate
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| show | bind subtrate pre-initiation complex of reaction, during transition state, and products after reaction is complete
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| What is that makes the high fidelity of DNA replication so surprising? | show 🗑
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| How do cyclic conformational changes in Pol I help it to achieve high fidelity? | show 🗑
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| How does the 3’ to 5’ exonuclease activity of Pol I help it to achieve high fidelity? | show 🗑
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| show | after replication, DNA is hemi-methylated (parental is, new is not) which indicates which strand may need reparation
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| show | pol operates 5'-> 3', so how can DNA repair both strands?
soln: semidiscontinous DNA synthesis: leading strand is polymerized smoothly and lagging goes 5'-3' in opposite direction in segments, so bubble can still move in the same direction
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| show | all DNAP requires a primer, but where does this come from?
soln: use RNA primers
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| show | with alpha32P labeled dNTPS and isolated fragments and exposed them to alkane hydrolysis which showed a ribonucleotide with 3' 32P (indicated RNA/DNA junction)
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| show | DNAP needs primer so it can be used, but ultimately must be able to replace/degrade it
leading strand only need this 1x, okazaki need for each fragment
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| What are the major components of the E. coli replisome and how do they work together to bring about semidiscontinuous DNA synthesis at a replication fork? | show 🗑
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| helicase role in E. Coli replisome | show 🗑
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| show | binds and stabilizes DNA in ss conformation after helicase melts it to prevent self pairing
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| primosome role in E. Coli replisome | show 🗑
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| show | the replicase --> main polymerase responsible for DNA polymerization in e. coli
polymerizes 5'-3' and edits 3'-5'
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| show | relaxes positive supercoiled DNA ahead of replication fork (topo I or II), topo II decatenates final product
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| DNAPI role in E. Coli replisome | show 🗑
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| show | joins okazaki fragments -- catalyzed ligation between last dNTPs (3'-5' of dNTPs)
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| show | 6 copies of a single polypeptide arranged in a spiral staircase with room in the middle for ssA-DNA where each subunit makes H bods to phosphates n 2 bp
achieves strand separation by a hand-over-hand mechanism
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| show | 1. hydrolyzes ATP and releases ADP which triggers the release of top subunit from adjacent
2. released subunit remains anchored by NTD
3. flexible linker between NTD and CTD allows released subunit to move down and clamp bottom subunit with ATP
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| show |
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| show | core: alpha, epsilon, sigma
sliding clamp: beta2
clamp loader: gama2, delta. delta'. psi, rho2
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| role of core modules in DNAPIII | show 🗑
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| show | increases processivity by dimerizing and forming clamp around DNA strand so it can accept B and A DNA
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| show | puts clamp on and links leading and lagging core polymerases
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| How are processivity and efficient recycling of Pol III at odds with one another? | show 🗑
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| How do the clamp and the clamp loader work together to ensure both processivity and efficient recycling? | show 🗑
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| show | DNAPI excises RNA primers by nick translation and DNA ligase seals nick to complete sequence
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| show | relax positive supercoils ahead of replication fork and decatenates final product
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| show | as strands separate, T gets lower and lower, so without topo, W would get larger and eventually movement would be prevented
topoII decatenates daughter chromosomes
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| show | DnaA recognizes and binds oriC and melts DNA to form replication bubble and then DnaB and DnaC can unwind to form pre-priming complex
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| In what ways do the initiation and elongation phases in eukaryotic replication resemble those of bacterial replication and in what ways are they different? | show 🗑
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| Why do eukaryotic chromosomes require multiple origins of replication | show 🗑
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| show | RNAH cleaves RNA duplex and poldelta and DNA ligase fills in an you end up with a fragment of DNA thats missing so telomerase serves as a template the RNA primer can be synthesized and the poldelta can resyn the missing
(otherwise cells die)
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| show | mutations in telomerase lead to premature telomere shortening (which means losing information) and ultimately leads to cell death
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| How do UV radiation lead to DNA damage? | show 🗑
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| show | any nucleophilic atom on DNA can be alkylated, sometimes leading to changes in base pair specificity
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| show | deamination -- replaces NH2 with O
oxidative: forms reactive oxygen species which can cause structural mutations or directly attack and break the DNA backbone
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| show | sometimes spontaneous C --> U deamination, and if U is supposed to be there, no way to check if it came from daminantion or if it's intentional
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| How does DNA damage affect the templating properties of the bases? | show 🗑
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| show | can remove methyl and ethyl by transfer; not an enzyme because gets irreversibly modified by reaction
--High levels of alkylation, alkylated AGT, act as a transcription factor to activate the gene that encodes
the DNA repair protein
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| what is AGT | show 🗑
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| show | nick translation:
in bacteria, the exonuclease and polymerase functions are both provided by polI through the process of nick translation -- size of gap can be 1-a few nucleotides
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| show | damaged base has a structural mutation causing it to flip out of normal helix, exposing it for recognition
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| show | 1. synthesize RNA is 5'-3' direction
2. require a DNA template
3. can synthesize RNA strands de novo
4. exhibit processivity
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| show | core can only initiate transcription randomly (from nicks/gaps), need holoenzyme for regular initiation
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| show | α2, β, β', ω
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| components of RNAP holoenzyme | show 🗑
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| show | anchors β, β' together
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| role of β, β' in RNAP core | show 🗑
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| show | inessential, but may be a chaperone for folding of RNAP
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| role of σ in RNAP holoenzyme | show 🗑
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| show | missing bands on gel showed where DNA did not get fragmented and showed where RNAP was bound to DNA
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| show | operon, -35 region, TATA (pribnow)/-10 region, initiation site
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| How are different factors used to direct core polymerase to different genes? | show 🗑
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| show | 1. closed complex formation
2. open complex
3. abortive initiation
4. promoter clearance
5. elongation
6. termination
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| What are the characteristics of the closed complex formation in the transcription cycle? | show 🗑
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| show | kinetically stable -- formation is essentially irreversible
RNAP is functioning like a helicase
(sig 54 requires ATP but sig 70 does not)
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| what are the characteristics of abortive initiation in the transcription cycle? | show 🗑
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| what are the characteristics of promoter clearance in the transcription cycle? | show 🗑
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| characteristics of elongation in transcription cycle? | show 🗑
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| show | signalled by GC rich inverted repeat because RNAP transcribes through palindrome and then pauses on formation of AU rich heteroduplex and there's no room for hairpin in the active site so transcript is released
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| How does DMS footprinting work and how was it used to demonstrate contact between RNA polymerase and the major groove and open complex formation? | show 🗑
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| What are the the primary and secondary channels and how are they involved in transcription? | show 🗑
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| show | beta and beta' act as zippers and moves the bubble with RNAP with metal ions
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| show | The Mg2+ ion is next to the last phosphodiester bond in the RNA
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| show | coordinate with amino acids and can deprotonate and create optimal bond geometry so the phosphoester bond is easier to form
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| show | σ domain induces DNA from -11 to +2 to melt and allow template strand in ssDNA region to enter active site and stay near metal catalyst
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| show | the σ 3-4 linke rregion blocks further initiation of transcript and leads to abortive transcripts -- once transcript can push linker out of the way, the σ factor is released and there is promoter clearance
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| what is the purpose of abortive initiation | show 🗑
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| show | blocks secondary channel -- tells us where NTPs enter
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| show | binds inside bubble and creates a steric clash after 2-3 residues
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| What is the role of Lac operon negative regulation? | show 🗑
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| show | 1. presence of lactose inactivates repressor
2. absence of glucose activates CRP which is an activator
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| show | It is different than the consensus sequence. It allows ensures the lac promoter is inactive in the absence of cAMP-CRP (Glucose). Positive regulation.
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| show | has more space between the backbones, so the bases are more exposed and more sequence information is available
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| How do HTH motifs recognize and bind specific DNA elements? | show 🗑
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| What is AR1 and how was it defined? | show 🗑
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| What is the recruitment model? (RNAP) | show 🗑
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| show | energetically favorable interaction between AR1 of CRP and CTD alpha subunit lends stability to RNAP and promoter
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| show | not required for transcription, but is required for CRP activation
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| How was it determined that the AR1 domain of CRP interacts with RNA polymerase? | show 🗑
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| what experiment showed that RNA polymerase interacts with CRP? | show 🗑
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| How were the eukaryotic RNA polymerases discovered? | show 🗑
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| show | RNAPI: class 1 genes
RNAPII: class II genes
RNAPIII: clas III genes
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| show | ~50% of cell's transcriptional activity and encodes 45s rRNA precursor
class I genes
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| show | ~10% of transcriptionala ctivity
transcribes genes encoding mRNAs and small RNAs
subject to explicit regulation
class II genes
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| show | accounts for 40% of cells transcriptional activity
transcribes genes encoding tRNAs, 5s rRNAs, and small RNAs
promoter is often internal to transcribed region
does class III genes
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| show | eukaryotic polymerases contain homologues of all bacterial core subunits --> structures f catalytic sites and mechanisms of catalysis are highly conserved. but eukaryotes contain additional subunits
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| What do class II core promoters look like? | show 🗑
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| show | binding site for regulatory factors and can be upstream or downstream from core promoter elements
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| show | = bacterial closed complex
contains TFIID, TFIIB, TFIIA TFIIF, TFIIE, TFIIH, Poll II and CTD
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| show | has ~15 subunits including a TATA binding protein (TBP)
TBP binds minor groove which must open to accomodate a 10-strand anti-parallel beta sheet
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| describe the TATA binding protein (TBP) of class II eukaryotic transcriptional machiner | show 🗑
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| show | as a kinase: CTD phosphorylation is linked to eukaryotic promoter clearance
as a helicase: open complex formation at class II promoters requires TFIIH as a helicase
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| What do we mean when we say that sequence transcription factors are modular in nature? | show 🗑
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| How do various types of DNA binding domains present recognition helices to the major groove? | show 🗑
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| What are three basic ways in which coregulators modulate rates of transcription? | show 🗑
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| What is the molecular nature of the Mediator? | show 🗑
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| where do dominant suppressors bind the mediator and what does this mean? | show 🗑
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| where do recessive suppressors bind mediator and what does this mean? | show 🗑
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| show | complex of DNA, histones, and non-histone proteins ound in the nucleus of eukaryotic cells
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| show | heterochromatin and euchromatin
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| what is the significance of heterochromatin | show 🗑
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| show | 'normal,' not condensed chromatin --> transcriptionally active during interphase and stains diffusely
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| show | chromosomes ar emade of condensed chromatin wrapped on nucleosomes
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| show | bead like structure on eukaryotic chromatin --> short length of DNA wrapped around core histone proteins
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| show | the CTD of core polymerases alpha subunit
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| show | the 8 histone proteins:
2 H2A-H2B dimers (a histone fold pair)
1 H3-H4 tetramer
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| show | pattern of post translational modifications on histones that can
influence gene function (activation or repression)
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| How do post translational modification result in gene activation and repression? | show 🗑
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| show | recognize acetylated lysines on histones --> protein sits on histone and LysAc interacts in the binding pocket near the bromodomain which forms H bonds
recognizes acetylated histones bc if no acetylation ==> no transcription
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| show | Gcn5, which is required for RNAPII is a HAT ==> Evidence that transcriptional machinery can acetylate histones
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| How is DNA methylation passed from one generation to another? | show 🗑
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| show |
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| show | 1. Exon shuffling: It is a process through which two or more exons from different genes can be brought together ectopically, or the same exon can be duplicated, to create a new exon-intron structure
2. Propagation of junk DNA - transposons
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| How do conformational changes in the spliceosome serve to bring the splice sites together and what is the role of ATP in these conformational changes? | show 🗑
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| show | 5' splice site, branchpoint, and 3' splice site
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| show | 2' OH group (nucleophile)
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| show | the C' epimer preferentially selected for the bulge loop. hydroxyl was not in right position (stereochemistry is wrong), so reaction does not occur
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| show | subbed hard base with a soft base and did not get final spliced complex
when removed metal ions (EDTA) no complex formation or splicing
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| show | holds the Mg for splicing rxn
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| show | structure: 7mGppp (7 methyl guanosine triphosphate)
purpose:
1. makes 5' end resistant against 5’ to 3’ exonucleolytic cleavage
2. helps ribosome recognize mature mRNA
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| show | ensures efficient channelling of transcript from transcriptional machinery ti capping machinery
(phosphorylated CTD is a docking site for capping enzymes)
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| What series of events leads to the formation of the mRNA 3’ end? | show 🗑
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| What is the purpose of the polyA tail? | show 🗑
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| What advantage does alternative splicing have over transcriptional regulation as a way of regulation gene expression? | show 🗑
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| How is splicing regulated in Drosophila sex determination? | show 🗑
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| What what is the role of guanosine in group I self splicing RNA? | show 🗑
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| Why do we think that the spliceosome is probably a remnant of the RNA world? | show 🗑
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| How was RNA interference discovered? | show 🗑
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| show | injecting sense and anti-sense and mixed RNAs into animals
saw that sense and antisense resulted in null phenotype but no null phenotype when using introns
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| show | The miRNA does not have complete complementarity to the target mRNA
-- siRNA has complementarity, but uses RISC to cleave to suppress transcription
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| How is the genetic code designed to minimize deleterious effects of mutations? | show 🗑
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| wobble hypothesis and why having a wobble base pair on a tRNA would be advantageous | show 🗑
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| show | have pseudouridine and dihydrouridine, both of which are dispensable -- unclear what purposes are
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| show | D-arm, T,pseudouridine,C arm, variable arm, and anti-codon arm
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| How does the cloverleaf fold up to form the L-shaped tRNA? | show 🗑
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| What is the purpose of tRNA charging? | show 🗑
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| show | attachment of amino acids to cognate tRNA, catalyzed by aminoacyl-tRNA synthetases
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| What are the two steps in tRNA charging? | show 🗑
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| How do aminoacyl-tRNA synthetases recognize their cognate tRNAs? | show 🗑
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| Why is editing required to ensure high fidelity tRNA charging and how does this editing work? | show 🗑
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| show | suggests ribosome is a ribozyme
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| How was the Yarus inhibitor used to prove that the ribosome is a ribozyme? | show 🗑
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| show | rate enhancement occured without change in activation enthalpy -- showed change occured via entropy increase as opposed to enthalpy decrease
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| show | via formation of duplex between s/d and 3' end of 16s rRNA to position start codon in P-site
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| Why are polycistronic messages common in bacteria, but not eukaryotes? | show 🗑
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| show | gtp-ases couple conformational changes to GTP hydrolysis and act as a motor to drive process of protein synthesis forwards
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| Chain elongation - How do GTPases drive aa-tRNA delivery and translocation? | show 🗑
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| show |
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| show | eukaryotic eIF-2 can be phosphorylated to form a dead end complex with eIF-2B and will be sequestered out of the cycle
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| show | HRI is inactivated by heme -- without heme can autophosphorylate eIF2 and sequester it out of cycle
GCN5 is activated by amino acid starvation and prevents inaccurate synthesis by sequestering out the eIF2 and preventing translation
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| What is the fundamental difference between the logic of the de novo purine and pyrimidine pathways? | show 🗑
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| Why do we have both salvage and de novo pathways? | show 🗑
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| why is the purine salvage pathway so important | show 🗑
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| Where does PRPP come from and what is the reason for using it in all nucleotide biosynthesis? | show 🗑
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| Pyrimidine biosynthesis | show 🗑
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| show | Salvage is a major pathway
Base synthesized while attached to ribose
IMP is common intermediate for AMP and GMP, but itself is not a typical nucleotide
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| Determination of the pathway - How was pigeon poop used to determine the metabolic sources of the purine ring atoms? | show 🗑
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| show | ammonia uses glutamine binding to channel for increased metabolic efficiency and avoids need to accumulate labile/toxic intermediates
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| What is the Ntn domain? | show 🗑
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| Why is it important to start out with the alpha anomer of ribose in the Amidophosphoribosyl transferase reaction? | show 🗑
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