Exam 2 review pt. 2

Question 1

What is the difference between transcription and translation?

Answer 1

Transcription is the process of reading the genetic code and producing
mRNA to transfer the code. Translation is the process of reading the
mRNA transcript and producing proteins that have some function in cellular
activities. Transcription takes place in the nucleus and involves DNA, RNA
Polymerases producing mRNA, whereas Translation takes place in the
cytoplasm and involves mRNA, Ribosomes, and producing peptides.

Question 2

What did the Beadle Tate experiment demonstrate?

Answer 2

The Beadle Tate experiment demonstrated that the genetic material of yeast could be mutated and that mutation would cause a defect in an enzyme (protein) function. The researchers reasoned that the genetic material could be linked to protein production by mutating it. The defect could be determined by screening the mutants for problems in using or metabolizing amino acids or nutrients. They did this by seeing which yeast cultures would die if they didn’t have a nutrient, assuming that if the mutant died without a nutrient that the mutation had something important to do either metabolism or use of the nutrient.
This is the basis for identifying the location of genes to a pathway and creating early gene maps.

Question 3

Draw a picture of a gene and its regulatory elements. Construct a Table
that identifies the DNA element and the corresponding protein interactant.

Answer 3

Enhancer Element- Activator
Silencer Element- Repressor
Regulatory TF Binding Sequences- Regulatory Transcription Factors
CpG Islands w/ methylation (gene repressed)-Methyltransferase, Methyl binding Proteins
TATA Box- TFIID
Transcription Start site- RNA Polymerase

Question 4

What is the protein that transcribes single gene DNA called?

Answer 4

RNA POL II (RNA polymerase II)

Question 5

Define the following terms as they relate to Transcription:
Template DNA, HN-mRNA, mRNA, Nascent strand.

Answer 5

Template DNA – The coding DNA strand that gets transcribed
HN – mRNA – Heteronuclear DNA – messenger DNA that has been made from transcription and is still in the nucleus (immature mRNA)
mRNA – RNA that has been made during trascription
Nascent Strand - The strand of mRNA during transcription which is actively being added to (growing mRNA strand from transcription)

Question 6

What are transcription factors, what types are there?

Answer 6

Transcription factors are proteins or elements that migrate to the gene that will be transcribed and up-regulate transcription by recruiting the RNA POL II polymerase to the start site.
Basal Transcription Factors – required for minimum transcription TFII D, A, B, F etc. work directly in assembling the Holoenzyme complex
Upstream Transcription Factors – bind to enhancer elements
Inducible Factors – bind to upstream elements but are controlled (activators and repressors)
Coactivators and Co-repressors – function to activate or repress transcription but not through direct DNA binding.

Question 7

Coactivators and corepressors interact with what to do what?

Answer 7

Coactivators interact with activators and repressors or with chromatin remodeling proteins HAT or HDAc to up or down regulate transcription.

Question 8

What is the difference between HAT (histone acetyltransferase) and HDAc (histone deacetylase). What is the chemical nature of the chromatin as it relates to acetylation of histones.

Answer 8

HATs transfer an acetyl group to the core histones to loosen the DNA-histone complex (chromatin) due to neg charge repulsion of the acetyl group to the phosphodiester bonds. HDAc removes the acetyl group from the histones which functions to compress the chromatin by interaction of Pos Charge Histidine and Lysine to Neg Charge phosphodiester bonds.

Question 9

What amino acids are rich in histones that attract the DNA?

Answer 9

Basic Amino Acids Lysine, Histidine and Arginine have positive groups that attract the negative charge of the phosphate group from the DNA strand.

Question 10

. What is the difference between euchromatin and heterochromatin which is likely to have hyperacetylated histones and which is likely to have hypoacetylated histones?

Answer 10

Euchromatin is loose – HYPER acetylated – pushes DNA away
Heterochromatin – tight HYPO acetylated – attracts DNA

Question 11

What type of proteins are involved in the Transcription Preinitiation complex formation, Name the 2 that make up the TFIID complex?

Answer 11

TATA Binding Proteins (TBP) and TATA Binding Protein - Associating Factors (TAFs) make up TFIID.

Question 12

What are the general properties of the Pre-initiation complex that allow transcription to take place.

Answer 12

Bend DNA
Interact with Major and minor grooves
Attract RNA Pol II to TATA site
Can act as coactivators
Melt DNA promotor elements
Helicase activity (DNA unwinding)

Question 13

What direction is the template DNA read and what direction is the growing mRNA chain made?

Answer 13

Read – 3’ to 5’
Made - - 5’ to 3’

Question 14

What is the term given to the process of growing the mRNA chain and what links the nucleotides together.

Answer 14

Elongation or polymerization, nucleotide are help together by the Phosphodiester bonds

Question 15

What may be some ways that the RNA POL stops transcribing the gene?

Answer 15

Pausing
Proof-reading
Repair
Termination Factors
Secondary structures of the nascent chain

Question 16

List the 3 events that occur to hnRNA to make it mature RNA.

Answer 16

5’ Methyl Guanine Cap addition, 3’ - Poly A Tail Addition, Splicing

Question 17

What is the purpose of the 5’ methylguanine cap and how does the Ribosome know that it isn’t just a regular nucleotide?

Answer 17

The 5’ methylguanine cap is a structure that identifies that the RNA is mRNA which needs to be translated. It recognizes eIF 4E in eukaryotes for translation initiation. Is involved in export out of the nucleus.

Question 18

What is the term half-life mean and how is it determined in the mRNA?

Answer 18

Half-life refers to the stability of the mRNA the longer the half life the longer the mRNA is in the cytoplasm for protein production. Technically the half-life is the time for one half of the mRNA to be left from the original value.
Probably controlled by secondary structure, polyA tail size, and other
factors.

Question 19

What mechanism acts at CG rich promoter areas to control gene transcription?

Answer 19

Methylation of the DNA promoter elements on the cytosine residues, block the binding or transcription factors.

Question 20

What experimental design would be used to show that a specific regulatory Transcription Factor binds to a specific promotor sequence?

Answer 20

EMSA or ChIP

Question 21

Explain the logic used to deduce that at least 3 nucleic acids must be needed to encode an amino acid during translation.

Answer 21

In order to get a combination that will encode for at least 20 amino acids you would need at least 3 nucleotides out of the 4 know to be present in DNA. 4^3 = 64

Question 22

What are 4 Features of the mRNA code?

Answer 22

Made of 3 nucleotides called codons
Non-overlapping
Degenerate (Redundant)
Wobble Property (Wobble base is on the tRNA)

Question 23

Explain how wobble and redundancy may protect against mutations or the effect of a mutation.

Answer 23

Redundant codons are often found having the same 2 first nucleotides in the code and the third which binds more loosely on the tRNA non-standard base. A non-standard base can bind weekly to all 4 standard bases and still carry the same amino acid it protects against mutation of the 3rd base in the codon.
An additional safe guard is in having similar amino acids with similar properties generally share the first 2 bases with the third base is different.

Question 24

You synthesis the following nucleotide sequences to determine the codons AAUAAUAAUAAU and UAUAUAUAUAUAU. What are the various codon combinations that could occur for each sequence?

Answer 24

AAU, AUA, UAA
UAU, AUA

Question 25

Define the following terms as they relate to Translation Initiation, IFs, Elongation, EFs, Ribosomes, rRNA, tRNA, mRNA and Nascent Strand

Answer 25

Initiation – Process of assembling the ribosome and first tRNA to the mRNA start site. IFs – Initiation Factors – proteins which assembly with the initiation complex and interact with mRNA sites, other proteins and/or the Ribosome. Elongation Process of linking the amino acids together to make a peptide chain. EFs – Elongation Factors – proteins that shuttle the tRNA to the Ribosomes during translation elongation and allow more the motor properties of ribosome. Ribosomes –Combinations of protein complexes with ribosomal RNA acting as a complex. rRNA – Ribosomal RNA – RNA sequences contained within the ribosome needed to recognize strand specific sequences on the mRNA. tRNA Transfer RNA – RNA structures that escort the amino acid associated with its anti codon to the mRNA during elongation mRNA Messenger RNA – RNA produced during transcription that is read during translation to produce the peptide Nascent Strand – The growing peptide strand made of linked amino acids.

Question 26

What 3 features of the prokaryotic mRNA designate the 5’ position and orient the translational start site?

Answer 26

Shine/Delgarno Sequence 5’ – Phosphate Group AUG Start Sequence

Question 27

What 3 features of the eukaryotic mRNA designate the 5’ position and orient the translational start site?

Answer 27

7-M Guanine Cap AUG Start Sequence Context Before AUG (GCCGCCACCACC)

Question 28

Where are the codon and anticodon located and what is similar about them?

Answer 28

Codon – mRNA AntiCodon – tRNA They are complementary to one another

Question 29

What is the function of the small ribosomal subunit vs the large ribosomal subunit in translation?

Answer 29

Small ribosomal subunit – Locates the start site and orients the direction of translation. The Small Unit then recruits the Large Unit to the right location/Orientation. The Large Subunit contains the enzymatic functional domains for translation.

Question 30

Describe Initiation in Prokaryotes.

Answer 30

Assembly of the 30S Pre-initiation Complex with IFs 1 & 3 Recognition and binding of 30S to Shine/Delgarno Site Assembly of the fMet – tRNA and IF2 Binding releases IF3 Binding of 50S SubUnit with hydrolysis GTP -> GDP Release of IF 1 & IF 2

Question 31

Describe Initiation in Eukaryotes.

Answer 31

Assembly of the 40S Pre-initiation Complex with eIFs 1A & 3 Assembly of the Ternary Complex (Met – tRNA, eIF2 and GTP) Assembly of the 40S Pre-initiation Complex with Ternary Complex to produce 48 S size Assembly of the mRNA Pre-initiation Complex (PABP, eIF4s ) Binding of 48S SubUnit to mRNA site (48S at this point) Addition of 60S subunit to 48S site at mRNA start site, hydrolysis of GTP -> GDP and release of eIFs

Question 32

What is the sequence of the anticodon of prokaryotic and eukaryotic initiating tRNA and what does it code for in each case?

Answer 32

Codon = 5AUG 3 Anticodon = 3UAC5 Prokaryotes – formyl-Methionine, Eukaryotes - methionine

Question 33

What 2 structures form independently and then come together during early eukaryotic translation?

Answer 33

Ternary Complex (tRNA, eIF2 and GTP) and 40 S Subunit +e IFs ( 1A & 3)

Question 34

During transcription we discussed how the eukaryotic mRNA is modified to make a mature mRNA. What features of eukaryotic mRNA interact with proteins in preparation of translation?

Answer 34

Methyl Guanine cap at 5’ end interact with eIFs PolyA Tail interacts with binding proteins PABP The PolyA tail and methylGcap come together from ionic forces between the eIF4 factors

Question 35

What are the features of tRNA and how does it get its characteristic shape?

Answer 35

tRNA has several stem loop structures made by localized regions of complementary base pairing. This forms a characteriatic “T” shape. The “t” shape is important in that it makes every tRNA the same relative size and structure so that the orientation and space between amino acids are the same during peptide bond formation. tRNA also contain non-standard bases in their structure. A non-standard base on the 5’ nucleotide of the anticodon can recognize more than one code at 3’ nt on the codon.

Question 36

What do the A, P, and E sites stand for in the translational complex, and what functions happen at each of these sites?

Answer 36

A- Aminoacyl Selection – this is the site where the acetylated - tRNA comes in and finds its codon P – Peptidyl Site – this site is where the peptide bonding occurs to link the growing peptide chain to the new amino-acid/tRNA entering at the A site E – Exit site - this is where the deacylated tRNA leaves the Ribosome and is recycled.

Question 37

What steps require energy for translation elongation and what process does the cell couple elongation with to get the necessary energy?

Answer 37

The delivery of the Amino-acetylated tRNA to the A site requires GTP-> GDP energy conversion The translocation of the Ribosome along the codon requires GTP-> GDP energy conversion.

Question 38

What does the term Reading Frame mean and explain how a mutation can arise from a shift in reading frames?

Answer 38

Reading Frame refers to the proper 3 nucleotides of the codon being read as a codon. A shift of one nucleotide backwards or forwards will change the code by altering the bases associated with the codons used for translation. A mutation can arise if the reading frame is shifted since different nucleotides will be read as a code leading to a different amino acid. A truncation can also result if a stop codon is the result of a frameshift.

Question 39

What are the 4 mechanisms that an antibody targeted to bacteria disrupts translation?

Answer 39

They can distort the 50S and cause incorrect codon reading They can prevent the tRNA from binding to the codon. They can interfere with peptide transferase and block peptide bonding. They can prevent translocation of the ribosome to the next codon.

Question 40

in what organism is transcription coupled to translation.

Answer 40

Prokaryotes

Question 41

What are the functions of the RER/Golgi as it pertains to protein translation and modifications?

Answer 41

Protein folding Addition of Carbohydrates Cleave peptide signals Assemble protein complexes Associated with vesicle transportation Anchoring additions (hydrocarbon chains, GPI anchors)

Question 42

Where are two locations for protein synthesis and what are the possible fates of these proteins upon their synthesis?

Answer 42

Cytosol and RER complex Cytosol – Proteins which function in the cytosol, nucleus, chloroplast, peroxisome, and mitochondrial proteins RER – membrane proteins, vesicle proteins, lysosomal proteins

Question 43

In general what is the mechanism through which a peptide gets directed to the membrane or to a particular organelle?

Answer 43

A signal sequence on the peptide itself directs it to a particular organelle or the RER. This sequence is recognized by chaperone proteins which direct recognition and/or entry by other proteins that are associated with the channel of the organelle.

Question 44

What are the steps in translation of a secretory protein?

Answer 44

Nascent peptide has a signal sequence that is recognized by signal recognition particle which directs the ribosome/peptide complex to the translocon. The ribosome/peptide/SRP docks at the translocon. There are 2 binding interactions SRP-SRPR and ribosome-translocon. GTP is hydrolyzed to free the signal peptide from the recognition particle and the SRP from the receptor. A conformational change widens the channel and the peptide is inserted through the translocon. It either goes all the way in or a stop-transfer anchor sequence signals translocation through the channel to make an integral protein. Multiple stop anchors and translocation events can make a variety of membrane spanning domains.

Question 45

What are 4 differences that could occur between different membrane bound (integral) proteins relative to their orientation in the membrane?

Answer 45

Size of the NH3 or COOH termini, location cytosolic or ectoplasmic face of either the NH3 or COOH termini, number of membrane spanning domains

Question 46

How is it possible to get the wide variety of different types of multi-spanning integral proteins in the membrane in the right orientations?

Answer 46

Two different proteins will have different signal sequences in different parts of the peptide chain for different events such as signal -anchor and stop-transfer anchor sequences throughout the peptide.

Question 47

What are the functions of the following structures or enzymes?

Answer 47

Signal Peptidase – cleave signal from peptide Protein Disulfide Isomerase – break and reform disulfide bonds Proteosomes – structure for degrading proteins – in particular defective or misfolded proteins from synthesis Glycosyltransferase – addition of sugar to saccharide chain Transamidase – transfer of a protein to a lipid making a GPI anchor

Question 48

What is the normal antero- directed progression of membrane protein sorting starting from the nuclear export of the mRNA? What proteins help direct these vesicles?

Answer 48

Antero – directed is from the RER to the cis Golgi to the trans Golgi and out to the membrane. COP II proteins transport from RER to cis Golgi while COPI proteins shuttle between the Golgi.

Question 49

Explain a model for membrane targeting of vesicles and their fusion to the ER membrane?

Answer 49

Currently it is viewed that vesicles are shuttled by adapter proteins (motor proteins) to tethering proteins located near the site of fusion. The fusion occurs from interaction of the vesicle SNARE proteins to the membrane SNARE proteins. At the vesicle/membrane interface Syntaxin and Complexin work with SNAREs to complete the Fusion-Pore opening.

Question 50

What are the steps in mitochondrial protein trafficking based upon whether the protein is needed as matrix protein or used between the membrane layers?

Answer 50

Peptide is formed in the cytosol and contains a mitochondrial (matrix) targeting sequence. The MTS is recognized by a Mitochondrial stimulating Factor (or by a heat shock protein). The peptide is unfolded and delivered to TOM receptor proteins that are assessory proteins to the TOM channel proteins. The protein is directed through the TOM channel and then into the TIM inner channel. The peptide is then directed to either the interspace membrane through translocation or reinsertion through a channel protein, or it remains in the matrix mitochondria.

Question 51

What is the major protein needed for both entry and exit from the nuclease. And what 2 proteins regulate GDP/GTP in nuclear transport ?

Answer 51

RAN is the major protein controlling entry and exit in nuclear protein trafficking. RAN has 2 binding conformations one for GDP and one for GTP. RAN/GDP is made outside by GTP hydrolysis controlled by RAN-GAP. Once RAN is in the GDP bound state it enters the nucleus. To release the protein needed it changes conformation, which releases the protein and GDP. RAN now has a conformation favorable for GTP binding. GTP binding to RAN is regulated by GEF (RCC1) in the nucleus allowing it to take proteins out of the nucleus when needed.