question

What do antibiotics inhibit?

answer

Bacterial translation in Eukaryotes, not affecting Eukaryotic RNA and protein function.

question

How is translation terminated?

answer

Stop codon, and Release factor that cause peptidyl transferase to catalyze a water molecule to the carboxyl end of the last amino acid

question

List the stop codons.

answer

UAA, UGA, UAG

question

Define an ORF.

answer

Open Reading Frame: series of codons starting with ATG and ending with a stop codon. Doesn't necessarily code a protein but possible.

question

Key features of a plasmid that allow it to be cloned:

answer

1. One of more useful restriction sites at which YFG can be inserted 2. Selectable marker that allows the identification of the plasid in bacteria 3. Orgin of replication to allow replication of plasmid, independently of host chromosome

question

What are the most common selectable markers for use in bacteria?

answer

Genes encoding resistance to antibiotics. These include resistance to ampicillin, tetratcycline, kanamycin and chloraphenicol.

question

What are the four steps of PCR?

answer

1. Denature DNA at 90-95°C 2. Anneal two primers ar 50-60°C 3. Extend the primers with a DNA polymerase and the 4 dNTPs at 72°C 4. Repeat steps 1-3 about 30 times

question

What is the number of molecules of YFG after n cycles of PCR?

answer

2ⁿ For example, after round 2 you would have 2² copies of YFG.

question

Which two technical advances have made PCR a general tool?

answer

Thermostable polymerases such as Taq polymerase and automated thermocyclers.

question

How do you seperate the different DNA fragments you got as a result of PCR?

answer

Seperate by size. Use gel electrophoresis in which DNA molecules can sieve through agarose/polyarcylamide. Small DNA molecules can sieve through pores in gel more easily, thus small DNA molecules will be able to sieve towards the positive end of the magnetic field faster.

question

Two routine methods for visualizing DNA on the gel in gel electrophoresis.

answer

Ethidium bromide between bases which will fluoresce red in UV light. ³²P to 5' ends of DNA fragment using gamma ³²P-ATP.

question

When using mRNA as the template _______ is often used as the primer for the synthesis of the cDNA as it will anneal to the polyA tail.

answer

poly-dT

question

Define Tm.

answer

Tm is the temperature at which 1/2 of a specific double stranded DNA has become single stranded.

question

Which can influence Tm?

answer

AT/GC content: The more GC content, the higher the Tm because 2 hydrogen bonds versus 3. Length of helix: Shorter molecules melt at lower temperatures. Salt content: Higher salt increases Tm becuase salt neutralizes the negative charge in the phosphate backbone. Solvent: Formamide and urea decrease Tm by weakening hydrogen bonds. Imperfect matching in DNA: Decreases Tm.

question

ssDNA can anneal with ____ and ____. RNA can anneal with ___.

answer

ssDNA, RNA. RNA.

question

Steps in a Northern Blot

answer

1. Isolate RNA from different tissues 2. Use gel electrophoresis to seperate RNA based on size 3. Transfer seperated RNA to a membrane (nitrocellulose/nylon) 4. Prepare radiolabeled single stranded complementary probe for fragment of interest 5. Incubate the probe with the filter at about 10°C below the Tm 6. Wash away any nonspecifically bound probe 7. Expose probed filter to X-ray film

question

When would you want to use Northern Blot?

answer

When you have a gene/RNA and you want to find out where it is expressed in other cell types or if it is expressed in other cell types. You probe DNA with RNA in Northern blots.

question

Restriction enzymes are Type ___ enzymes. Which show specific ____ and specific _____ of DNA.

answer

II, recognition, cleavage

question

What is a palindromic sequence?

answer

A sequence that reads the same both ways.

question

When can you have sticky ends and when can you not?

answer

5' overhang and 3' overhangs result in sticky eneds that can base pair and reanneal. Blunt ends cannot base pair and reanneal and thus do not have sticky ends.

question

The DNA ligase enzyme is usually isolated from what?

answer

T4 bactriophage

question

Do DNA ligase require energy to reseal compatible sticky ends?

answer

Yes, given a 5' phosphate DNA ligase will reseal compatible sticky ends using ATP.

question

Can DNA ligase reseal blunt ends?

answer

Yes, but less efficently.

question

What are some difficulties assosciated with expression of eukaryotic proteins in bacteria?

answer

-Incorrect folding possibility -Lack of post-translational modification (phorsphorylation, glycosylation) -Poor Stability

question

What is glycosylation?

answer

Post-translational modification through the attachment of sugars to proteins.

question

Why is site directed mutagenesis used?

answer

To produce proteins that have improved or special qualities becuase the gene that was used to produce them was mutated. Also used to create a protein that has lost its function because of an altered gene sequence.

question

State the steps of site directed mutagenesis.

answer

1. Oligonucleotide with desired mutation will anneal with single stranded template at the position to be altered 2. dNTPs and DNA polymerase extend the oligonucleotide forming a second strand 3. DNA ligase will seal the nick and the result will be a double stranded template **Can then use PCR to amplify DNA to allow for cloning**

question

What are DNA microarrays and when do you use them?

answer

DNA microarrays are a way of analyzing thousands of genes at a time. The standard use of microarrays is to find out what genes are expressed in a cell under certain conditions.

question

How do you use DNA microarrays?

answer

Two conditions, two cDNA probes per condition. One tagged with red fluor and other with green fluor. The two fluorescent probes are hybridized to an array with DNA with all or many ORFs in a cell type and detected by a laser microscope. Red spots indicate genes that are only expressed in condition X, Green spots indicate genes that are only expressed in condition Y and Yellow spots indicate genes that are expressed in both conditions.

question

What is RNA profiling?

answer

cDNAs produced randomly from cell tissue and sequenced. The number of reads of the sequence is proportional to the level at which it is expressed at that random time.

question

How are Short Tandem Repeats used for DNA fingerprinting?

answer

STRs vary in individuals. To fingerprint an individual you use primers around the STRS and then amplify the STR by PCR. Then you can use gel electrophoresis to analyze becuase different STRs will run at different positions on the gel.

question

What linkage is formed between sugars in DNA and which carbons are involved in the bond?

answer

Phosphodiester linkages; from the 5'carbon to the 3' carbon

question

What is the significant difference between the sugar in DNA and RNA?

answer

DNA does not contain an Oxygen atom on the 2' carbon hence the term "deoxy-"

question

Whats the difference between purines and pyrimidines?

answer

Purines consist of two rings fused together whereas pyrimidines are only one ring

question

What tautomer of the nitrogenous bases is most prominent in solution?

answer

Keto form; more stable *keto form and enol form have different hydrogen bonding capabilities thus one is favoured over the other*

question

Which one of the following statements regarding the DNA double helix structure is FALSE? a) the structure is antiparallel (5' of one strand adjacent to 3' strand of the other) b) the helix is left handed c) the sugar backbone is on the outside, and the

answer

B) the helix is RIGHT handed

question

Why is the sugar backbone found on the outside of the structure?

answer

Phosphates of sugar backbone have a negative charge, and thus by facing outwards the negative charge is spread out

question

How many hydrogen bonds for between A-T? G-C?

answer

2 for A:T 3 for G:C

question

How thick is a DNA strand?

answer

2 nm

question

What is the distance between base pairs?

answer

0.34 nm

question

How many base pairs in one complete turn of DNA?

answer

10

question

Why is there a minor and major groove on the the DNA helix?

answer

Bases are not symmetrically oriented relative to the backbone.

question

In alkaline solution is DNA more stable or is RNA?

answer

DNA; RNA tends to degrade in alkali solution becuase of it's 2'-OH group that interacts with the base.

question

What is the main difference between the structure of Uracil and Thymine?

answer

Thymine has a methyl group at where a uracil has a hydrogen. Otherwise, rest of the structure is the same.

question

True or False: RNA is usually longer than DNA

answer

False, DNA is longer.

question

True or False: RNA and DNA are both sequence dependent structures

answer

False, DNA is not sequence dependent as it is always a double helix.

question

True or False: DNA is less likely to have its nitrogenous bases modified in comparison to RNA

answer

True, RNA is more likely to be modified.

question

True or False: RNA can exist as single stranded or double stranded.

answer

False, RNA is single stranded yet it can from intramolecular bonds with itself to form unique structures.

question

What is "chromatin"?

answer

Complex of DNA and its organzing proteins.

question

What are "histones"?

answer

Histones are proteins that make up chromatin, they are used to condense DNA.

question

Which of the following statements about histones are FALSE? a) composed of positively charged lysine and arginine residues b) each histone is composed of fewer than 200 amino acids c) found in prokaryotes and eukaryotes d) all of the above are true

answer

C, histones are only found in eukaryotes!

question

What does it mean when the sequence for a histone is "conserved"?

answer

The sequence for histones are very similar in various organisms indicating that the histone have been evolutionarily conserved. Also, that histones are very important since they have been conserved over evolutionary time.

question

What is an "octamer"?

answer

Consists of 4 histone proteins (H2A, H2B, H3, H4) to make a DNA packaging complex. Two of each histone proteins make up histone octamer(8).

question

How many times does a DNA wrap around an octamer?

answer

1.7 times (147 bases)

question

Which histone is not found in an octamer?

answer

H1, it is instead used for adjacent linking of nucleosomes.

question

What is a "nucleosome care particle"?

answer

Histone octamer and DNA wrapped around it.

question

What is a "nucleosome"?

answer

Nucleosome core particle plus adjacent linker DNA to next nucleosome. Linkers can be 3-80 bp long.

question

By what factor do nucleosomes compact DNA? a) 3x b) 5x c)10x

answer

3x

question

Approximately how many base pairs can be found withing one nucleosome?

answer

200 base pairs!

question

What structure do nucleocomes fold into?

answer

30 nm chromatin fibres

question

What histone stabilizes the 30 nm chromatin fibres?

answer

H1

question

What vague structure do 30 nm chromatin fibres fold into? How many base pairs are found in each structure?

answer

Loops, 30-200 kbp in one loop.

question

What complex allows DNA to decompact for reading?

answer

Chromatin Remodeling Complex/ Chromosome Remodelling Complex

question

Given a nucleotide sequence 5' - ATGTACCTG - 3', what is the complementary strand? a) AGTTCGTGC b) ATGTACCTG c) TACATGGAC d) CAGGTACAT

answer

D: 3'-CAGGTACAT-5'

question

What is a "chromosomal scaffold"?

answer

System of non-histone proteins that support the 30 nm chromation loops structure (3rd level of folding).

question

True or False: Chromosomal remodelling complexes require ATP to unfold DNA

answer

True, Chromatin Remodelling Complexes require ATP

question

What is a poorly understood factor related to histone structure/folding?

answer

Histone chemical modification that can involve methylation, phosphorylation, acetylation

question

Which of the following is the CORRECT order of DNA replication steps? a) initiation → priming → DNA synthesis → proofreading → ligation b) priming → initiation → proofreading → DNA synthesis → ligation c) ligation → initiation → priming → DNA synthesis → DNA cleavage d) initiation → priming →proofreading → DNA synthesis → ligation

answer

A, initiation → priming → DNA synthesis → proofreading → ligation

question

Where does initiation occur?

answer

At the replication origin. A richness in TA base pairs indicates an initiation site. Single replication origin found in prokaryotes, but multiple replications through prokaryote genome.

question

What protein is responsible for unwinding the DNA double helix?

answer

DNA Helicase

question

What did Meselson and Stahl discover about DNA replication and what was their theory called?

answer

Semi-conservative theory - upon replication each new daughter strand consists of a parent strand and a newly synthesized strand.

question

What is the function of "inititaion proteins"?

answer

To recognize replication origins and pull apart the two strands.

question

What base pairs are most common in the replication orgin and why?

answer

TA base pairs because they are relatively weaker then CG base pairs which share 3 hydrogen bonds.

question

Which binds to the replication origin first, the initiator proteins or DNA Helicase?

answer

Initaior proteins

question

What does DNA Helicase require to unwind the DNA?

answer

ATP

question

What prevents the two DNA strands separated by DNA Helicase from reannealing?

answer

Single stranded binding proteins help hold the two strands apart.

question

True or False: Following the unwinding of DNA, DNA Polymerase binds to each template strand and begins the synthesis of a new strand.

answer

False, DNA polymerase cannot begin replication without primers

question

What is the function of "primase"?

answer

To lay down primers (~10 nucleotides) on the DNA strand which will be extended 5'-3' from their 3' end by DNA polymerase. The primers provide DNA polymerase with a starting pointing.

question

In which direction does DNA Polymerase synthesize the new strand onto the template strand?

answer

5'-3' on the NEW strand. (But, 3'-5' relative to the template strand.)

question

What protein is responsible for keeping DNA polymerase on the template strand?

answer

Sliding Clamp Protein

question

What are "Okazaki fragments" and where are they found?

answer

Okazaki fragments occur on the lagging strand becuase DNA synthesis occurs in a discontinuous manner on the lagging strand.

question

True or False: The lagging strand requires multiple primers, whereas the leading strand generally requires only one

answer

True, the lagging strand synthesizes discontinuously and thus it requires many primers for the start of each Okazaki fragment

question

What nitrogenous bases are pyrimidines?

answer

Cytosine, Thymine, Uracil

question

True or False: The leading strand is synthesized first, and the lagging strand is synthesized second

answer

False, both are synthesized concurrently

question

What protein connects Okazaki fragments together?

answer

DNA ligase

question

True or False: Many replication bubbles form along a DNA strand during replication

answer

True (in eukaryotes)

question

True or False: Primers in DNA replication are composed of RNA.

answer

True

question

What binds to the newly synthesized DNA strand once DNA Polymerase encounters an RNA primer and exits replication?

answer

Another DNA polymerse (DNA polymerase I)

question

What is 5'-3' exonuclease activity?

answer

It is when DNA polymerase proofreads as it is synthesizing, looking for and removing errors such as deletion, insertion or substitution.

question

How often do DNA replication errors occur in humans?

answer

once every 10⁷ bases

question

True or False: DNA synthesis and 5' to 3' exonuclease activity occur at different active sites on DNA polymerase

answer

True

question

Apart from proofreading, what other function does 5' to 3' exonuclease activity have?

answer

Removal of RNA primers that were formed at the start of replication as the new DNA is being synthesized.

question

What two things does DNA ligase require to connect DNA fragments into one strand?

answer

ATP (humans) and NAD+(prokaryotes) A 5' phosphate group and 3' OH group

question

What product is formed when DNA Ligase hydrolyzes ATP?

answer

AMP (adenosine monophosphate)

question

What is the function of tRNA?

answer

To carry amino acids to the ribosome allowing for translation

question

What is the difference in function between DNA Polymerase III and DNA Polymerase I?

answer

DNA polymerase III synthesizes new DNA until it hits the next RNA primer where it disasssociates. DNA polymerase I comes in and removes the RNA primer, synthesizing DNA in its place.

question

What are telomeres composed of?

answer

Proteins and DNA that contains many repeats.

question

Approximately how long is a telomere?

answer

5 000-15 000 bp

question

True or False: The 5' end of a telomere hangs off and invades the DNA earlier in the telomere sequence

answer

False, it is the 3' end of the telomere that hangs off and invades.

question

How long is the overhang of a telomere?

answer

30-200 bp

question

Why do telomeres contain many repeats?

answer

Allows for the 3' overhang to form a loop with a earlier segment in the telomere. Also, as chromosomes divide DNA is lost at the ends of the chromosome. Telomeres act as buffers at the end of chromosomes to provide safety to the chromosome such that when it divides no critical information is lost, only repeat sequences are lost.

question

True or False: Telomeres contain gene sequencing DNA.

answer

False

question

State the two purposes of telomeres.

answer

1. Allow chromosomes to distinguish between a natural true end of a chromosome versus a double stranded break 2. Provide extra length at the ends to prevent gene coding DNA from being lost due to inabailty to synthesize DNA to the very end at the lagging strand

question

Can the lagging strand be synthesized to the chromosome tip? If not, why not?

answer

No, it cannot becuase the final RNA primer doesn't go to the very end of the lagging strand.

question

What protein is responsible for the synthesis of telomeres? Does this protein extend the template strand or new strand?

answer

Telomerase extends the template strand on the lagging strand, and then an RNA primer is added again to the template lagging strand and DNA polymerase synthesizes the rest of the complementary strand. http://faculty.plattsburgh.edu/donald.slish/Telomerase.html

question

True or False: Telomerase expression in somatic cells stops shortly after birth.

answer

True

question

How does the expression of telomerase relate to cancer cells?

answer

Telomerase is continuously expressed in cancer cells, preventing them from dying becuase of lost genetic information.

question

How does AZT combat HIV?

answer

AZT mimicks a sugar that would be incorporated into the viral DNA, however the "AZT sugar" does not contain a 3'-OH preventing the viral DNA from being able to elongate.

question

What group is found on the 3' carbon in AZT instead of a hydroxyl group of a normal sugar?

answer

An azide group: N₃

question

True or False: Viral polymerase incorporates AZT into DNA, whereas human polymerase does not.

answer

False, human polymerase does incorporate AZT into DNA but just very infrequently. This is the reason that causes people taking AZT to feel sick.

question

What "copying mistakes" can occur in DNA?

answer

Deletion, insertion, substitution

question

What is depurination and when does it occur? What impact does depurination have on DNA replication?

answer

Spontaneuous loss of a G or A. This usually promoted by acid. When a base is lost, DNA replication is stalled since theDNA polymerase doesn't what tp do when a base is missing.

question

What results when depurination remains unrepaired?

answer

Deletion in the daugher double strand where the G/A was found.

question

How can replication continue past the site of depurination?

answer

When DNA polymerase falls off becuase it was stalled, Translesion DNA polymerase comes in and either inserts a random base in the place of G/A or it skips the missing base and replicates past the site of damage. Translesion DNA polymerase works on a few sites but then the original DNA polymerase comes back and continues replication because the error-prone Translesion DNA Polymerase is too risky.

question

What is deamination?

answer

Deamination is when a amine group spontaneously converts into a carbonyl. This most commonly happens to cytosine, converting the cytosine into a uracil.

question

What is the result of unrepaired cytosine deamination?

answer

The U links with an A in the mutated daughter double strand. The G that original C was bonded to turns into a A in the mutate daughter double strand. Eventually you end up with a T linked to a A in the double strand.

question

What are Pyrimidine Dimers?

answer

Formation of a cyclobutane ring between two adjacent pyrimidines becuase of exposure to UV light. Most frequently, thymine dimers are the result. Dimers block replication.

question

How can replication continue past the site of thymine dimers?

answer

Translesion DNA polymerase can overcome the block by synthesizing DNA past the site of mutation or incorporating a mutation in the place of the dimer.

question

What can result in breakage of sugar-phosphate backbones or chemical changes in bases?

answer

Radiation, Chemical mutagens (O₂⁻ superoxide radical), mechanical stress

question

What can be the result of double strand breaks?

answer

Chromosome abnormalities or cell death.

question

How is the newly synthesized strand of DNA identified in bacteria?

answer

Bacteria add methyl groups to their bases in the DNA over time, so a newly synthesized strand would not have methyl groups on bases.

question

How are mismatch errors corrected in bacteria?

answer

- Endonuclease nicks backbone where mismatch is - Exonuclease goes in and removes the error - DNA polymerase goes in and fills in the removed DNA correctly

question

How is the newly synthesized strand of DNA identified in eukaryotes?

answer

The newly synthesized strand in eukaryotes tends to have nicks in the sugar phosphate backbone. Origin unknown but a proposed theory is that DNA polymerase sometimes adds in RNA while synthesizing which are removed, resulting in nicks.

question

How are mismatch errors repaired in eukaryotes?

answer

- mismatch repair proteins look for nicks in the newly synthesized strand - Exonuclease degrades the newly synthesized strand from the point of the nick to the point of the mismatch - DNA polyermase and DNA ligase come in and resynthesize using other strand as template

question

Mismatch repair only fixes mistakes made by DNA _________.

answer

Polymerase!

question

What errors are fixed by Base Excision Repair (BER)?

answer

Localized, small types of damage such as modified bases, abasic sites and single stranded breaks.

question

What are the 5 steps of BER?

answer

1. Base excision by exonuclease 2. Endonuclease makes a nick in the sugar phosphate backbone to indicate to DNA polymerase the right position for synthesis 3. DNA polymerase adds the correct base, resulting in a overhanging sugar phosphate backbone 4. The extended sugar phosphate backbone is gotten rid of by endonuclease 5. DNA ligase comes in and seals the nick in the backbone.

question

What is the difference between short patch BER and long patch BER?

answer

Short patch BER only excises 1 nucleotides whereas long patch BER can excise 2-10 nucelotides.

question

What errors are fixed by Nucleotide excision repair?

answer

Major damages such as large chemical damage to the DNA double helix resulting in DNA damage that is broader than a single base. ex. repair of thymine dimers that disrupts double helix

question

What are the four steps of NER?

answer

1. Endonuclease nicks the backbone at either side of damaged section (10-30 bp apart) 2. DNA helicase removes the damaged section 3. DNA polymerase resynthesizes removes section 4. DNA ligase seals the sugar phosphate backbone

question

What two methods of DNA repair fix double stranded breaks?

answer

Homologous and Nonhomologous recombination.

question

What is the process of homologous recombination?

answer

Damaged DNA molecule matches up with an undamaged DNA molecule to ensure accurate repair using undamaged DNA as template.

question

What is the process of nonhomologous recombination?

answer

DNA ligase quickly joins the two broken ends of the double strand break regardless if information is loss.

question

What is the key difference between homologous and nonhomologous recombination?

answer

Loss of information.

question

When does homologous recombination occur more frequently than nonhomologous?

answer

In organisms with smaller genomes since homologous recombination is a slow process requires easy acces to undamaged DNA molecule. Homologous recombination tends to occur shortly after replication since it requires a template double strand to match to.

question

What are the three reasons why DNA is transcribed into RNA?

answer

1. Allows for gene amplification 2. Can be easily degraded providing mechanims to turn genes off 3. Provides additional oppurtunities for modification

question

How many protein encoding genes are found in a human cell?

answer

About 21 000 different protein encoding genes.

question

How many points of gene regulation exist in a eukaryotic cell?

answer

7

question

Which of the following is NOT a method of gene regulation during transcription? a) making RNA at different quantities b) phosphorylating/acetylating the RNA strand c) terminating transcription of RNA at different points d) elongating the RNA at different speeds

answer

B, phosphorylating/acetylating is done during protein processing

question

How are genes regulated during RNA processing?

answer

- 5' 7-methylguanosine capping - 3' polyadenylation - RNA splicing - RNA editing

question

True or False: RNA processing occurs within and outside the nucleus.

answer

False, RNA processing occurs only before the RNA is transported outside of the nucleus into the cytoplasm of the cell.

question

How are genes regulated during mRNA export?

answer

Proteins in the nuclear pores highly regulate what mRNAs can leave the cell. They must have 5' 7-methylguanosine cap, 3' polyA tail, cap-binding protein, polyA binding protein and exon junction complex to exit the cell.

question

True or False: mRNA degradation in the cytoplasm of the cell occurs randomly and is controlled by the amount of copies of the same RNA strand produced

answer

False, mRNA degradation is a higly regulated process by RNA induced silencing complexes made up of microRNA and specialized proteins.

question

In the three steps of translation, which is the most regulated in the cell? a) initiation b) elongation c) termination

answer

a) Initiation AUG codon can be repressed by hairpins or by translation repressor proteins.

question

In which two ways is protein expression regulated within the cell?

answer

Protein modification: proteolytic cleavage to activate a protein, phosphorylation, glycosylation, acetylation, methylation and ubiquitylation Protein Degradation: amount of protein present in a cell is dependent on the balance of rate of synthesis and rate of degradation

question

What step in the biological process is most regulated and why?

answer

The first step because it saves energy that would need to be used unnecessarily to stop further steps in the process.

question

What is the first step of gene expression?

answer

Initiation of Transription

question

What is the function of a promoter?

answer

To initiate transcription. Promoter seqeunces recognize RNA polymerase and any gene specific regulatory factors.

question

What is the function of a terminator?

answer

To terminate transcription. Terminator sequences tell RNA polymerase to fall off.

question

What is an operon?

answer

An operon consists of genes on a single strand of RNA transcribed by a single promoter. NOTE: operons are a component of prokarytotic transcription

question

If gene X and gene Y in bacterial DNA are to be translated, what is the CORRECT order of sequences 5' to 3' on the DNA strand? a) promoter → operon [gene X → gene Y] → terminator b) promoter → gene X → promoter → gene Y → terminator c) terminator → operon [gene Y → gene X] → promoter d) terminator → gene Y → promoter → gene X → terminator

answer

C, transcription is read 3'-5' on the template strand for transcription to occur 5'-3' 3' - terminator → operon [gene Y → gene X] → promoter - 5'

question

What other sites apart from the promoter are essential for transcriptional initiation in prokaryotes?

answer

the -10 and -35 sequence

question

What is the -10 and -35 consensus sequence?

answer

-10: TATATT -35: TTGACA

question

How far apart are the -10 and -35 sequences?

answer

about 16-19 bp apart

question

Why are the -10 and -35 promoter sequences known as "consensus" sequences?

answer

Not all promoters have the same sequence, but as an average consensus at -10 the promoter sequence is TATATT and at -35 it is TTGACA. The consensus sequence is made up of the most frequently occuring bases at the sites in functionally related DNA elements.

question

What protein is responsible for synthesizing RNA transcripts?

answer

RNA polymerase

question

What does RNA Polymerase use as substrates to synthesize RNA transcripts?

answer

NTPs (nucleotide triphosphates)

question

What's the difference between NTPs, dNTPs, and ddNTPs?

answer

NTPs are used in synthesizing RNA. They contain the sugar ribose. dNTPs are used in synthesizing DNA. They lack a 3'-OH and are made up of deoxyribose rather than ribose. ddNTPs are used to terminate replication. They lack a 2'OH and 3'OH. Because of the missing 3' OH, DNA extension is not able to continue and replication is terminated. They are useful in genetic analyses.

question

What are the two forms of RNA polymerase?

answer

1. Core enzyme: RNA polymerase alone 2. Haloenzyme: RNA polymerase + sigma

question

What is the difference between transcription by a core enzyme and a holoenzyme?

answer

Core enzyme doesn't recognize promoters, it just synthesizes RNA at nicks in DNA only. Haloenzyme recognizes promoters. NOTE: DNA primase is a type of RNA polymerase, probably the core enzyme.

question

What structural differences exist between the core enzyme and haloenzyme of RNA polymerase?

answer

Core enzyme has 4 subunits: 2 α and β and β'. Haloenzyme has 5 subunits: 2 α, β, β' and a sigma subunit.

question

What is the purpose of the sigma subunit?

answer

The sigma subunit recognizes the -10 and -35 promoter sequences. Different sigma subunits exist to recognize different promoter sequences. When genes have better promoter sequences, they bind haloenzymes better and result in more transcription. For example, σ⁷⁰ is the housekeeping sigma σ⁵⁴ is for nitrogen metabolism σHS is for heat shock

question

True or False: Sigma factors are a transcription initiation factor in eukaryotes.

answer

False, sigma factors enable specific binding of RNA polymerase to gene promoters in prokaryotes.

question

What is a closed complex?

answer

RNA polymerase haloenzyme bound to promoter.

question

What is an open complex?

answer

Sigma subunit has left, RNA polymerase alone unwinds strands at the promoter.

question

Are primers required for RNA synthesis?

answer

No.

question

What four substrates are used in RNA synthesis? What kind of nonds are formed between these substrates? What is released when a bond is formed?

answer

ATP, CTP, GTP, UTP Phosphodiester bonds are formed. Pyrophosphate/diphosphate is released.

question

When does a sigma subunit fall off? And when does it bind to RNA polymerase again?

answer

Sigma subunit falls off 5-10 nucleotides after the promoter. It binds with RNA polymerase again when RNA polymerase reaches the terminator sequence and fall off.

question

True or False: The transcription bubble moves downstream with RNA ligase reannealing the template DNA behind.

answer

False, the DNA template needs to reanneal to its complementary strand, not single stranded RNA.

question

What are 2 methods of regulating how much of a certain gene is transcribed in prokaryotes?

answer

1. Strength of -10 and -35 sequences for binding to sigma subunits 2. Regulatory proteins such as activators and repressors

question

What does the Trp operon code for?

answer

5 enzymes involved in synthesis of tryptophan

question

What is the mechanism of the tryptophan repressor?

answer

When low tryptophan, RNA polymerase is recruited to the tryptophan promoter in -10 and -35 sequences allowing for transcription. When high tryptophan, Trp repressor binds to tryptophan to be activated, and it binds to operator site within the tryptophan promoter and blocks transcription for anymore tryptophan.

question

How does the Trp Repressor binds to DNA?

answer

Trp repressor undergoes a conformational change when tryptophan binds to it which allows it to form a dimer with DNA. It makes base specific contacts with DNA using helix 5 of its helix turn helix motif.

question

How does binding of the Trp repressor and Tryptophan prevent RNA Polymerase from undergoing RNA synthesis?

answer

Binding of the repressor sterically inhibits access of RNA polymerase to the promoter. It specifically prevents the sigma subunit from locating the -10 and -35 sequences.

question

How many amino acids are in the Trp repressor?

answer

107 amino acids

question

Which of the following makes up the structure of the Trp repressor? a) two alpha subunits, and 4 alpha helices b) 3 alpha subunits, 3 alpha helices c) 4 alpha helices, and two beta subunits d) 6 alpha helices

answer

D: 6 alpha helices makes up the Trp repressor.

question

Is the lac operon subject to negative or positive regulation in E. Coli?

answer

Both.

question

What two signals are required for Lac operon to function?

answer

Absense of glucose Presence of lactose

question

Tryptophan is a _____ protein. CAP is an _________ protein.

answer

Repressor, Activator

question

What occurs in Lac operon during absence of glucose and presence of lactose?

answer

cAMP levels rise which binds to CAP causing a conformational change, and CAP drives the digestion of lactose. CAP binding site is upstream(5') of the -35 sequence and through direct protein-protein interactions it recruits RNA polymerase to the promoter. Allolactose binds to Lac Repressor which makes the repressor release from operator allowing for the transcription of LacZ which breaks down lactose into glucase and galactose. OPERON ON

question

What occurs in Lac operon during presence of glucose and absence of lactose?

answer

Low cAMP levels, CAP not activated and this RNA polymerase is not recruited. Lac repressor remains bound to promoter preventing transcription of LacZ. OPERON OFF

question

What occurs in Lac operon during presence of glucose and lactose?

answer

cAMP levels low, so CAP does not bind and does not recruit RNA polymerase. Presence of lactose cause Lac repressor to fall off. OPERON OFF

question

What occurs in Lac operon in absence of glucose and lactose?

answer

cAMP levels rise, CAP binds to its binding site upstream of the -35 sequence but it does not recruit RNA polymerase becuase the Lac Repressor remains bound to the promoter.

question

True or False: The Trp and Lac Repressors both bind at a promoter in between the -10 and -35 promoter sequences.

answer

False, the Lac repressor binds to a site downstream the -10 promoter sequence but the Trp repressor binds to the Trp promoter located in the -10 and -35 sequences.

question

What is responsible for positive regulation in Lac operon?

answer

Absence or presence of glucose

question

What is catabolite repression?

answer

When glucose is present, the expression for genes required for the metabolism of other carbon sources is limited.

question

True or False: When glucose levels are low, cAMP levels are also low.

answer

False, when glucose levels are low, cAMP levels are high.

question

What is the difference between RNA polymerase in eukaryotes versus prokaryotes?

answer

Prokaryotes have one type of RNA polymerase. Eukaryotes have 3 different RNA polymerases.

question

What are the three different RNA polymerases in eukaryotes and what is each used for?

answer

RNA poly I: rRNA RNA poly II: mRNA RNA poly III: tRNA and snRNA

question

What does it mean when gene transcription is "monocistronic" and where does this transcription occur?

answer

One gene = one promoter Monocistronic genes are found in eukaryotic cells.

question

State the differences in the promoter region of eukaryotic and prokaryotic cells.

answer

Eukaryotic cells have a TATA box found upstream of the +1 transcriptional start site, they do not have -10 and -35 sequences nor do they have sigma subunits.

question

What complexes does RNA Polymerase require to bind to the promoter region in eukaryotic cells?

answer

TATA binding protein which is a compenent of the TFIID transcription factor.

question

What is the main difference in regulatory protein binding sites between prokaryotes and eukaryotes?

answer

Regulatory binding sites are generally way further upstream from promoter than in prokaryotics.

question

What 2 methods are used to change the structure of nucleosomes?

answer

1. Chromosomal remodelling complex 2. Enzymes such as histone acetyltransferases and histone deacetylases

question

True or False: RNA processing occurs at the same time as transcription

answer

True

question

State the 3 reasons why a 5'cap is required.

answer

Required for: 1. RNA export out of nucleus 2. Stabilizing the mRNA from being degraded 3. Acting as a Translation initiation signal

question

What is a polyA tail and how long is it?

answer

300 repetitive adenine bases added to 3' end of mRNA. PolyA tail is added after mRNA is cleaved after approximately 30 bases post the AAUAAA sequence. The polyA tail provides additional stability to mRNA and prevents it from being reduced by 3' exonucleases.

question

In what way does Trp repressors act as a "dimer"?

answer

Two monomers of the Trp repressor bind together creating a symmetric dimer structure which is required for inhibition of trypotophan promoter.

question

What is spliced out of mRNA to produce mature mRNA?

answer

Introns

question

What is "differential splicing"?

answer

Ability to put exons together in many different ways resulting in production of various proteins. This accounts for the large coding capacity of the genome.

question

What 3 sequences are required for splicing to occur?

answer

5' junction, branch point, 3' junction

question

What base is vital for splicing in the branch point?

answer

Adenine

question

What type of reactions occur when splicing out introns?

answer

2 Transesterification reactions

question

What is the first step of intron splicing?

answer

2'OH from adenine in branch point attacks 5' junction which releases the 5' junction from the intron. Lariat formed.

question

What is the second step of intron splicing?

answer

3'OH from 5' splice junction attacks phospjhatr group of 3' splice junction creating a bond and releasing the lariat structure.

question

What is a splicesosome/snRNP?

answer

snRNAs and additional proteins

question

What is the role of snRNPs: U1, U2, U4, U5, U6?

answer

U1 binds to 5' splice site U2 binds to lariat branch point Additional snRNPs are attracted to the splice site and they further excise the intron leaving the spliced mRNA behind.

question

In what way does RNA have catalytic potential?

answer

It can catalyze its own conversion to mRNA.

question

Where are snRNPs not required for pre-mRNA?

answer

In protozoa, mitochondria and chloroplasts

question

What is a key transcription factor responsible for positive regulationin yeast?

answer

Gal4

question

What is the UASgal?

answer

Upstream Activating Sequence - galactose which is a unit consisting of 4 sites on the Gal10 promoter which are bound to Gal4.

question

When is Gal4 active?

answer

In the presence of galactose

question

When galactose levels are low, how is GAL4 inhibited?

answer

By the repressor protein Gal80 which keeps Gal4 in its inactive state.

question

How does GAL4 become active in the presence of galactose?

answer

Galactose + Gal3 to Gal80, freeing Gal80 from Gal4 allowing to transcribe.

question

True or False: The GAL genes regulate transcription through protein-nucleic acid interactions

answer

False, the Gal genes interact through protein-protein interactions.

question

During catabolite repression, what transcription factor is responsible for the negative regulation of the GAL genes?

answer

Mig1 binds to Gal10 promoter region in presence of glucose inhibiting transcription.

question

How does Mig1 inhibit transcription?

answer

Mig1 recruits factors that modify chromatin in the vincinity of Gal10 to inhibit transcription

question

What happens to Mig1 when glucose is not present?

answer

It is phosphorylated and cannot enter the nucleus

question

Mig1 is used for _____ regulation of Gal genes. Gal4 is used for _____ regulation of Gal genes.

answer

Negative, Positive.

question

True or False: Gal4 does not alter chromatin structure

answer

False, Gal4 stimulates transcription by protein-protein interactions with components of transcriptional machinery and by altering chromatin structure.

question

What compenents allow for RNA export?

answer

5' 7-methylguanosine cap, 5' cap protein, exon junction complex, 3' PolyA tail, PolyA binding protein, proteins in nuclear membrane

question

What motif does Gal4 form?

answer

Zinc finger

question

Gal4 recruits RNA Polymerase ___.

answer

Gal4 recruits RNA Polymerase II.

question

Exons = expressed, Introns = ______.

answer

Interruptions

question

What comes first, the initiation proteins or primase?

answer

Initiation proteins

question

What is a "codon"?

answer

Three base sequence that codes for an amino acid or signal. Ex. AUG is the start codon

question

How many codons does the cell need?

answer

20 as there are 20 amino acids, but there are many more than that.

question

How many possible codons are there?

answer

4³ = 64 different codons because 4 different bases possible in one of the three different positions in a codon.

question

What does it mean when the genetic code is described as "universal"?

answer

The same codons coding for the same amino acids are found in all organisms, although there is codon bias in organisms.

question

What does it mean when the genetic code is described as "non-overlapping"?

answer

The codons are seperate and consecutive, non-overlapping.

question

Why does overlapping not occur in the genetic code?

answer

It would result in limitations in what amino acids could follow each other, becuase the last base of one codon would have to act as the first base for another.

question

Why did the genetic code evolve to not have any gaps?

answer

Gaps would cause the genetic code to be unnessarily long and complex by taking up space that serves no function.

question

How are gaps in the code removed?

answer

Splicing during mRNA processing

question

What is a "wobble" and where does it occur?

answer

Redundancy at one of the three bases in a codon still codes for the same protein, most frequently occurs at the 3rd position of the codon.

question

What does it mean when the genetic code is described as "commaless"?

answer

There are no gaps between codons, they are sequential.

question

Proteins/Amino acids that are needed more tend to have more _____ such that if a silent mutatuon occurs the needed protein can still be made because redundancy.

answer

Codons

question

Why do related amino acids have similar codons?

answer

Increased chance of a functional protein in the case of a single base mutation.

question

What is a missense mutation and what causes it?

answer

Difference in a single base that results in a different amino acid

question

What is a frameshift mutation and what causes it?

answer

Insertion or deletion of bases resulting in an altered amino acid sequence past the site of mutation.

question

What sort of frame mutation is the least likely to impact the rest of the sequence?

answer

An insertion or deletion of exactly 3 bases.

question

What is a nonsense mutation?

answer

A type of missense mutation that results in a premature stop codon.

question

What is a common shape for all tRNA?

answer

Cloverleaf/Stem-loop structure

question

What are the two key regions of tRNA?

answer

- Anticodon - short single stranded region at 3' end with amino acid that matches codon

question

What is the sequence at the short single stranded region at the 3' end on tRNA that allows it to bind amino acid?

answer

CCA

question

What bond is formed between the acceptor site of tRNA and amino acids?

answer

High energy ester linkage formed between amino acid and tRNA by tRNA synthetase.

question

What is the shape of tRNA in 3D?

answer

L-shape

question

True or False: tRNA can base pair with more than one codon.

answer

True

question

What is required for a tRNA to bind to a codon?

answer

Antiparallel base pairs with at least 2 positions of the codon (Wobble).

question

What are the 2 functions of aminoacyl tRNA synthetase?

answer

1. Binds amino acid to tRNA 3' CCA site 2. Providing specificity by ensuring correct amino acids bind to correct tRNA anticodon

question

How many different aminoacyl tRNA synthases are found in the cell?

answer

20, one for each amino acid

question

What is the general structure of ribosomes?

answer

Large subunit: 49 proteins, 3 RNAs Small subunit: 33 proteins, 1 RNA

question

What are the 3 binding sites within a ribosome and what are their functions?

answer

A site, aminoacyl tRNA site. P site, peptidyl tRNA. E site, exit site.

question

How many binding sites can be occupied at one time in a ribosome?

answer

2

question

In which direction does the ribosome decode the mRNA strand?

answer

5' to 3'

question

How is translation initiated in eukaryotes?

answer

- Intiator tRNA with methionine amino acid loaded onto small subunit with initiator factors - Loaded small subunit recognizes 5' cap and moves 5'-3' to find AUG codon - Inititaion factors dissociate, large subunit comes in and binds to small subunit - Initiator tRNA repositions to P site

question

How is translation initiated in prokaryotes?

answer

- Ribosomes recognize binding sequences upstream from each functional AUG - Prokaytic ribosomes readily bind to start codon - RNA-RNA base pairing allows for ribosome recognition

question

What is the second step of elongation in translation?

answer

Energy from the ester linkage of tRNA in P-site is used to link amino acid in A-site to amino acid in P-site

question

What is the first step of elongation in translation?

answer

Charged aminoacyl tRNA comes into the A site.

question

What is the third step of elongation in translation?

answer

Large subunit translocates forward (5'-3'), moving tRNA in P site → E site, and tRNA in A site → P site.

question

What is the fourth step of elongation in translation?

answer

Small subunit translocates forward (5'-3'), ejecting tRNA in E site. 1 tRNA still remains in P site.

question

What are the reasons why bacteria cannot initiate translation in the way eukaryotes do?

answer

Prokaryotes do not have a 5' cap and they have polycistronic genes unlike monocistronic genes in eukaryotes.

question

What is an "adenoma"?

answer

Benign tumour (can be surgically removed)

question

What is an "adenocarcinoma"?

answer

Malignant tumour (has spread elsewhere in the body)

question

What is "metastasis" and how many cancer cells survive it?

answer

Movement of cancer cells from one spot to other parts of the body. 1 in 1000 cancer cells survive metastasis.

question

What are the 3 steps of metastasis?

answer

1. Cancer Cells grow as benign on Basal Lamina 2. Cancer Cells pass through Basal Lamina into blood stream 3. Cancer Cells leave blood stream and make their way to a new tissue

question

How do we know Cancer is a genetic disease?

answer

Certain cancers are inheritable (Recall: Brca1, Brca2) DNA damage can cause cancer (UV light)

question

What are two preventable causes of cancer?

answer

Environmental cues (smoking, radiation) Viruses (HPV)

question

What percentage of cancer cases are linked to smoking? What percentage of lung cancer can be attributed to smoking?

answer

30%, 90%

question

What is the relationship between incidence of cancer vs age? Why so?

answer

Exponential chance of cancer as you age because over time you acquire more mutations within cells of your body.

question

True or False: Cancer is caused by a single mutation

answer

False, it takes an accumulation of mutations to lead to cancer (min. 10)

question

True or False: Cancer cell have an infinite lifespan

answer

True, they don't respond to signals that would stop cell division

question

What are the four properties of cancer cells?

answer

1. Immortal 2. Genetically Unstable 3. Can grow in abnormal places 4. Divide in absense of growth factors

question

How do cancer cells proceed through the cell cycle for division?

answer

They skip over all the checkpoints that would normally stop a defected cell from dividing.

question

What are 2 types on cancer causing genes?

answer

Oncogenes and Tumour supressor genes

question

What is an "oncogene"?

answer

Mutant form of a normal gene that experiences gain of function allowing it to act dominantly and cause cancer.

question

What is a "proto-oncogene"?

answer

Normal gene that can become an oncogene. (for instance, a mutation can cause it to divide rapidly)

question

What are the 3 types of genetic change that can lead to an oncogene?

answer

1) Sequence mutation leading to hyperactive protein made in normal amounts 2) Gene amplification 3) Chromosomal rearrangement of a gene in front of a very active promoter

question

What is a "tumour supressor gene"?

answer

A gene who's absence causes cancer

question

What is the most common tumour supressing gene?

answer

p53

question

Is apoptosis triggered in cancer cells?

answer

Nope.

question

What are the two nonspecific forms of treating cancer?

answer

Radiation and Chemotherapy

question

What is Gleevac and what does it treat?

answer

Treats Chronic Myeliod Leukemia It prevents the binding of Bcr-Adl by acting as a competitive inhibitor to ATP which is needed to bind the two. The binding od Bcr-Adl causes CML.

question

True or False: There is no tRNA for stop codons.

answer

True, a release factor comes in rather than a tRNA when a stop codon is reached.

question

Base stacking involves what types of bonds/interactions?

answer

Van der waals interactions

question

If a DNA polymerase lost its 3'-5' exonuclease activity, how would the mutant polymerase compare to the normal?

answer

To replicate the same amount of DNA, the mutant would hydrolyze fewer dNTPs than the normal polymerase.

question

Which protein is most amount at a replication fork?

answer

SSBP

question

(Transcriptional/Translational) control is a key step in bacterial gene expression.

answer

Translational control is a key step in bacterial gene expression, as transicriptional cannot be as controlled because of the prescence of operons.

question

Do bacteria have mitochondria?

answer

No, prokaryotes do not have mitochondria or chloroplasts.

question

What is a limitation on the use of PCR?

answer

The sequence at the beginning and end of the DNA to be amplified must be known.

question

Where do you want your restriction enzymes to cut to allow you to insert your cDNA into a plasmid and allow its expression?

answer

You want to cut at your start codon and past the stop codon to coporate both as they are essential for your ORF.

question

What is true about the expression of human proteins in bacteria?

answer

Protein can be produced in high abundance, can be easily purified, may be insoluble, can possibly be modified correctly.

question

How do you read a chain termination sequence based of gel electrophoresis?

answer

Bottom(5') to Top(3')

question

In a ribonucleoside, which sugar carbons are directly bonded to an oxygen?

answer

1', 2', 3', 4', 5'

question

If a chromosome within a cell has a lower degree of packing or is less ordered, what is the most likely explanation?

answer

Genes in that section are being actively expressed

question

How long are Okazaki fragments in humans?

answer

100-300 bp

question

What comes first, primase or initiator proteins?

answer

Initiator proteins

question

At a replication bubble how many leading and lagging strands are there?

answer

2 leading, 2 lagging

question

How many bases are replaced during normal mismatch repair, in humans?

answer

Varies, could be up to a couple hundred.

question

What interaction is required for the recruitment of RNA polymerase to the promoter in the inititaion of transcription?

answer

Protein to DNA interaction

question

Can RNA polymerase unwind DNA by itself? What about DNA polymerase?

answer

RNA polymerase can unwind the DNA strand, DNA polymerase requires DNA helicase.

question

Do the bases in the major groove look different than the bases in the minor groove in DNA from the point of view of a binding protein?

answer

Yes.

question

The nuclear membrane is an ______ structure with a _____ bilayer.

answer

Asymmetrical, bilayer

question

Does protein exit the nucleus?

answer

Yes.

question

What source of energy drives the coupling of tRNAs?

answer

Hydrolysis of ATP

question

What substrate is used to form peptide bonds in proteins?

answer

aminoacyl tRNAs

question

How does a ribosome select which aminoacyl is correct for the A site?

answer

Ribosome determines there is base pairing between the codon on mRNA and anticodon in tRNA.

question

In eukaryotes, approximately where is the ribosome binding site/SD box located?

answer

approx. 8 bases 5' to each AUG

question

Amount of staining of ethium bromide is ______ to mass.

answer

Proportional

question

What type of nucleic acid can be used to probe a Southern Blot?

answer

RNA or ssDNA

question

How can you remove RNA from an RNA-DNA duplex?

answer

Alkali or RNase H

question

Which sugar carbon are bases attached to?

answer

1'

question

It is diffifult to obtain DNA sequence beyond 700-800 bases, why?

answer

Lack of signal after this number of bases and resolution of bands is difficult.

question

True or False: In prokaryotes translational initiation requires a ribosome binding site just upstream the AUG codon, but not in eukaryotes?

answer

True

question

What do eukaryotes have in place of a ribosomal binding site for translation initiation?

answer

TATA box

question

To clone Your Favourite Gene, where would the two primers for PCR anneal?

answer

Directly at the start codon of the gene including the AUG codon and downstream the stop codon.

question

What property of the cells does ampicillin affect?

answer

Cell wall synthesis, unlike other antibiotics which typically inhibit translation.

question

How do you prevent plasmids from reannealing after they have been cut by restriction enzymes?

answer

Remove 5' phosphates from plasmid.

question

Can mutations be inserted through PCR?

answer

Yes, primers are error prone.

question

What is the first step of protein purification?

answer

Lyse the cell.

question

How are sequence gaps possible?

answer

Because we originally sequenced both ends of some clones

question

True or False: Cancer originates from a single cell.

answer

True

question

Formaldehyde and Benzene are found in cigarette smoke, what effect do they have on DNA?

answer

Formaldehyde denatures DNA/RNA Benzene is a strong mutagen

question

What activates and inactivates Gal4?

answer

Prescence of galactose

question

How is Gal4 activated?

answer

Gal3 binds Gal80 to remove Gal80 off Gal4, stimulating transcription of Gal10.

question

How does Gal4 stimulates transcription?

answer

Protein-protein interactions and alteration of chromatin structure.

question

What is the purpose of Mig1?

answer

Catabolite repression: glucose presence causes the limation of expression og gene required for other carbon sources.

question

How does Mig1 inhibit transcription of Gal1o?

answer

Mig1 binds to Gal1o promoter.

question

True or False: The phosphorylated form of Mig1 inhibits transcription.

answer

False, the phosphorylated form of Mig1 cannot get into the nucleus to inhibit transcription.

question

Gal80 is a _______ protein.

answer

repressor

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