Oucod Biochemistry Exam 4 Levine

Front 1

Two fundamental processes involve DNA and two involve RNA in bacteria and mammalian cells:

Back 1

DNA: Replication and transcription. RNA: Transcription and translation

Front 2

three important features of the structure of RNA:

Back 2

1. Single stranded 2. Alkali-labile 3. Has uridine instead of thymidine

Front 3

What are the 4 requirements of DNA dependent RNA polymerase?

Back 3

a) Core enzyme b) DNA strands, c) Mg & Zn ions, d) NTPs

Front 4

The bacterial RNA polymerase core enzyme has 4 subunit polypeptides,what are they?

Back 4

alpha, beta, beta' and omega.

Front 5

What is the function of the alpha subunit of the bacterial RNA Polymerase core enzyme?

Back 5

assembly

Front 6

What is the function of the beta subunit of the bacterial RNA Polymerase core enzyme?

Back 6

catalysis

Front 7

What is the function of the Beta' subunit of the bacterial RNA Polymerase core enzyme?

Back 7

binding to template

Front 8

What is the function of the Omega subunit of the bacterial RNA Polymerase core enzyme?

Back 8

chaperone

Front 9

What is the function of the sigma cofactor used with bacterial RNA Polymerase core enzyme?

Back 9

Sends the core enzyme to the promoter, a sequence upstream of the region of DNA to be copied

Front 10

Why is the bacterial enzyme with sigma factor referred to as a holoenzyme, not the core enzyme?

Back 10

Because sigma factor dissociates when the enzyme is catalyzing the synthesis of RNA

Front 11

In what direction does RNA polymerase extend a chain? What metal ion is essential for catalysis.

Back 11

The new chain grows from 5’ to 3’because the previously attached ribose 3'-OH group attacks an incoming nucleotide triphosphate, which adds to that end. Catalysis requires Mg+2 ions

Front 12

What is a TATA box and why is it not transcribed?

Back 12

RNA polymerase only transcribes from the template strand downstream of where sigma-70 or TBP binds. Downstream of the TATA box on the coding strand is where the two strands become separated by helicase action of the holoenzyme

Front 13

What is the immediate result of successful promoter function?

Back 13

Separation of the two DNA strands within the RNA polymerase holoenzyme

Front 14

How do the sigma and rho proteins function?

Back 14

Sigma binds promotor sequences on DNA to initiate RNA synthesis. Rho binds CA rich terminator RNA sequences and this stops RNA synthesis

Front 15

Indicate three ways in which the process of transcription is more complex in eukaryotes than in prokaryotes?

Back 15

a) Different polymerases for different types of RNAs b) Many additional accessory protein factors c) Core enzyme has additional C-terminal domain (CTD) that gets phosphorylated

Front 16

Describe three characteristics of a prokaryotic mRNA transcript that differentiates it from a eukaryotic transcript?

Back 16

Prokaryotic mRNA transcripts have no introns, are not processed and are translated immediately because they are rapidly degraded

Front 17

Actinomycin D inhibits transcription in (some/all) organisms.

Back 17

ALL. Actinomycin D inhibits all RNA transcription because it binds to certain (GpC) sequences in DNA, preventing the open transcription complex from forming.

Front 18

Rifampicin binds to the active site of (bacterial/eukaryotic) but not (bacterial/eukaryotic) RNA polymerase

Back 18

Rifampicin binds to the active site of bacterial but not eukaryotic RNA polymerase

Front 19

alpha-Amanitin binds strongly to RNA Pol II, inhibiting (eukaryotic/bacterial) but not (eukaryotic/bacterial) mRNA transcription

Back 19

alpha-Amanitin binds strongly to RNA Pol II, inhibiting eukaryotic but not bacterial mRNA transcription

Front 20

What is a gene?

Back 20

A gene is an unbroken section of DNA that is transcribed to RNA and nearly always encodes a protein

Front 21

What is an exon?

Back 21

Exons are the regions of mRNA that are expressed (translated) into a protein.

Front 22

What is an intron?

Back 22

Introns are intervening regions of the gene that must be excised before the gene is translated.

Front 23

Assuming that siRNA and miRNA are subsets of scRNA, name the other 4 RNA types in a eukaryotic cell and order them from greatest to least amounts present. Where is scRNA in this ordered group?

Back 23

rRNA > tRNA > mRNA > snRNA. scRNA would be last, after snRNA

Front 24

Which of these RNAs is absent from prokaryotes: rRNA, tRNA, mRNA, snRNA? Why?

Back 24

snRNA, because there are no introns to be processed in bacterial mRNA

Front 25

Freshly synthesized eukaryotic mRNA is processed at 3 sites. One site utilizes a complex of three 3 enzymes, one a complex of two enzymes and one a complex containing a single enzyme activity. What are the sites, and the alterations to the mRNA that results?

Back 25

Sites are the 5’-end, the 3’-end and the middle: i.e. the cap, polyA tail and intron excision

Front 26

What enzyme component acts at the cap?

Back 26

Phosphohydrolase, guanosyl transferase and methylase of the cap synthesizing complex

Front 27

What enzyme component acts at the tail?

Back 27

Endonuclease and polyA polymerase of the poly A polymerase complex

Front 28

What enzyme component acts at the intron?

Back 28

Endonuclease formed by snRNA particles of the spliceosome complex

Front 29

What part of what enzyme do two of the three complexes (of the freshly synthesized mRNA that is being processed) attach before acting?

Back 29

C-terminal domain of eukaryotic RNA pol II

Front 30

How does each complex (used in the processing of freshly synthesized eukaryotic mRNA) recognize the portion of mRNA that it acts on?

Back 30

Cap synthesizing complex recognizes the 5’-end triphosphate ; The endonuclease of the polyA synthesizing complex and the snRNA particles of the spliceosome complex recognize consensus sequences on the unprocessed mRNA

Front 31

What are the functions of exon-intron 5’ and 3’ boundaries and of a downstream adenosine residue near the intron’s 3’end?

Back 31

Consensus sequence nucleotide residues that signal an intron to be excised from freshly synthesized mRNA in the nucleus. The downstream adenosine residue is also involved in splicing out the intron

Front 32

What nucleotide is the substrate for the first endonuclease action of the spliceosome complex? What are the spliceosome products in eukaryotic cells?

Back 32

An adenylate residue within the intron being excised. Products are processed mRNA with the exon removed plus the excised intron in the form of an RNA lariat

Front 33

How would you determine whether a given genes is being expressed by a mammalian or other eukaryotic cell at any given time and/or in a given environment?

Back 33

The proteome is not expressed all at once and all synthesized proteins must have an mRNA available to initiate translation. It has a poly-A tail that can hybridize with poly-U or poly-dT (The procedure of hybridization is not discussed in the handout). The mixture of eukaryotic mRNAs can then be probed with part of the nucleotide sequence that encoded a specific protein. If the probe hybridizes with the mRNA mixture, the protein is likely to have been expressed.

Front 34

What enzymes are required to process freshly synthesized tRNA and rRNA?

Back 34

Specific endo- and exo-nucleases that trim the ends and may also remove an intron without requiring the snRNA spliceosome complex

Front 35

One gene can encode two different polypeptides. How?

Back 35

1) Alternative splicing 2) Limited proteolytic processing

Front 36

What are the functions of the cap and tail in eukaryotic mRNAs?

Back 36

The cap facilitates splicing. The tail facilitates mRNA transport out of the nucleus . Both facilitate the initiation of translation by attaching to the ribosomal small subunit . Cap and tail are also the sites of initial endonuclease mRNA degradation

Front 37

In eukaryotic cells, where are ribosomes synthesized?

Back 37

Ribosomes are made in the nucleolus of the nucleus

Front 38

In eukaryotic cells, where are proteins synthesized?

Back 38

proteins are made in the cytosol, often attached to the rough endoplasmic reticulum

Front 39

Why is the genetic code composed of trinucleotide sequences and not dinucleotide or tetranucleotide sequences?

Back 39

Dinucleotides have only 16 possible combinations, not enough to encode 20 different amino acids. Tetranucleotides have 256 possible combinations, far too many possibilities

Front 40

With what was polyU incubated and how did that indicate its function?

Back 40

It was incubated with the alkali-treated cytosolic fraction of either E coli, or liver cytosol that contained polyribosomes (microsomes; 20,000 g supernatant). The sediment was radioactive only if the mixture was incubated with radioactive phenylalanine

Front 41

How were specific sequences of nucleotides shown to encode specific amino acids prior to elucidating the genetic code?

Back 41

Various trinucleotides were incubated with ribosomes purified from the alkali-treated cytosolic fraction of E. coli. Amino acids bound depending on the trinucleotide composition used

Front 42

How is amino acid activation for polypeptide synthesis accomplished?

Back 42

Activation is achieved by amino acid synthetases that bind ATP, a specific amino acid and a specific tRNA. Reaction produces pyrophosphate (PPi) AMP and aa-tRNA

Front 43

Why are there two classes of amino acid tRNA synthetases?

Back 43

Enhance synthetase specificity for the correct amino acid

Front 44

Why is the genetic code not overlapping?

Back 44

There would be insufficient room for aligning the aa-tRNAs next to each other to induce peptide bond formation if codons overlapped

Front 45

To what does the ‘wobble’ hypothesis apply and why is it necessary?

Back 45

It applies to mRNA/tRNA codon/anticodon interactions on the ribosome during protein synthesis It is necessary to limit the number of tRNAs because most amino acids are specified by more than one codon

Front 46

What are ribosomes and how are they categorized/measured? Hint: S

Back 46

Ribosomes are particles composed of structural RNA (no protein encoding function) and protein made in the normal way. The sizes of ribosomal particles or their component RNAs are measured in Svedbergs, a sedimentation coefficient measurement

Front 47

What enzyme synthesizes eukaryotic ribosomes?

Back 47

The DNA genes encoding eukaryotic ribosomal RNA are transcribed by RNA polymerase I

Front 48

How many tRNAs are needed to translate all 4 codons of glycine, GGX where X is any of the 4 nucleotide bases? How many different amino acyl transferase enzymes would be required? Explain.

Back 48

A minimum of 2 tRNAs but only 1 glycine-specific transferase enzyme. There is inadequate wobble at the third codon base to allow one tRNA to fit all 4 codons. However, there may be 2, or 3 or 4 tRNAs depending on the anticodon sequence of the tRNA. Only 1 transferase is required because all tRNAs with a given amino acid specificity are structurally indistinguishable to the enzyme that transfers that amino acid

Front 49

What are the 4 main features of a tRNA molecule

Back 49

1. amino acid attachment arm 2. dihydrouridine (DHU) arm 3. pseudouridine arm 4. anticodon arm

Front 50

What is the function of the amino acid attachment arm on a tRNA molecule?

Back 50

recognized by amino acyl synthetase AND peptidyl synthetase

Front 51

What is the function of the dihydrouridine (DHU) arm on a tRNA molecule?

Back 51

sandwiches over the pseudouridine loop when changing from cloverleaf to L-form

Front 52

What is the function of the anticodon arm on a tRNA molecule?

Back 52

interacts with mRNA

Front 53

What is the significance of the tRNA L form?

Back 53

tRNA L form spreads out the tRNA across the ribosome, connecting the complementation to mRNA on the small subunit with the peptidyl synthetase reaction on the large subunit

Front 54

What are the 5 main steps of protein synthesis?

Back 54

1) Amino acid activation 2) Initiation 3) Elongation 4) Termination, 5) Polypeptide modification, folding and processing

Front 55

How does each ribosome assemble with mRNA and tRNA so that translation can proceed?

Back 55

A 30S ribosomal subunit binds to the mRNA by the Shine-Dalgarno nucleotide sequence, and then an f-met-tRNA binds to the AUG codon to form a 30S complex. The 50S ribosome subunit then attaches to form a 70S complex ready for elongation

Front 56

What are the functions of the three prokaryotic initiation factors?

Back 56

IF1 and IF3 bind to the small ribosome subunit. IF1 blocks a second (acceptor) site used for elongation and IF3 prevents the large subunit from attaching until after f-met-tRNA binds. IF2 contains GTP and binds to f-met-tRNA. When the large subunit binds, a GTPase hydrolyzes the GTP, causing IF2 to fall away as the 70S complex forms

Front 57

Initiation in eukaryotes differs from that in prokaryotes in 4 major ways. What are they?

Back 57

a) The 40S subunit binds the mRNA cap and tail b) The 40S subunit slides along the mRNA to the AUG codon using a helicase (no Shine-Dalgarno nucleotide sequence) c) Methionyl tRNA initiating (met-tRNAi) is not formylated d) More initiating factors than in prokaryotes

Front 58

Indicate the 4 major steps of elongation:

Back 58

1) Proof-reading 2) Delivery 3) Peptide bond formation 4) Translocation.

Front 59

What major cofactors are associated with the steps of proofreading and delivery in the process of elongation?

Back 59

EF-TS, EF-Tu and GTP are associated with proofreading and delivery.

Front 60

What major cofactors are associated with the step of translocation in the process of elongation?

Back 60

EF-G and GTP are associated with translocation

Front 61

Peptidyl synthetase is a ribozyme and has (multiple/no) cofactors

Back 61

Peptidyl synthetase is a ribozyme and has no cofactors

Front 62

How does EF-G•GDP interact with the ribosome to cause translocation?

Back 62

EF-G•GDP stabilizes the twisted form of the ribosome. Once bound, it exchanges its GDP for GTP. Hydrolysis of the GTP to GDP translocates the ribosome to the post-translocation state

Front 63

What is unique about the codon for transcription termination?

Back 63

Signaled by termination codons that bind protein release factors instead of an amino acyl tRNA

Front 64

Calculate the ATP equivalents (used as GTP or ATP) needed to synthesize a polypeptide during translation.

Back 64

Two ATPs are required to activate (make aa-tRNA), 1 GTP/aa for initiation or delivery and 1 GTP for translocation or termination. Total is 4 ATP or equivalents for every amino acid present in the polypeptide

Front 65

What are the functions of the ribosomes during protein synthesis? There are 6!

Back 65

a) Initiates translation by 30S subunit binding to Shine-Dalgarno sequence or eukaryotic mRNA cap b) Channel for mRNA to interact with aa-tRNA c) GTPase on its 50S subunit to ensure initiation, delivery, translocation and release d) GDP/GTP exchange factor for translocation and release e) Peptidyl transferase (a ribozyme) in the large subunit f) Tunnel in the 50S subunit for the peptide to escape

Front 66

Which of the following is not an antibiotic: Erythromycin (an aminoglycoside), Tetracycline (or Doxycycline), Cycloheximide, Puromycin, Streptomycin, Chloramphenicol? Why not?

Back 66

Cycloheximide. It binds only to a eukaryotic ribosome. Because it does not inhibit bacterial protein synthesis, it cannot affect bacterial growth and is therefore not an antibiotic

Front 67

Name one antibiotic that affects each of the following processes during prokaryotic protein synthesis: translocation, delivery, initiation and peptidyl synthetase?

Back 67

a) Erythromycin (aminoglycoside) b) Tetracycline c) Streptomycin d) Chloramphenicol

Front 68

What causes ribosomes to become attached to the rough endoplasmic reticulum? What is the result?

Back 68

Presence of an N-terminal signal peptide that attaches SRP to the ribosome. Polypeptide is moved out of the cytosol via a translocon

Front 69

All eukaryotic proteins have methionine at their N-terminus residue, but when secreted proteins are purified they usually do not begin with methionine. Why not?

Back 69

Secreted proteins have an N-terminal signal sequence that is cleaved on secretion. Thus, only intracellular proteins, for example hemoglobin, retain methionine at the N-terminus.

Front 70

What are the 5 compounds required for N-linked glycoside synthesis and where does this process mostly occur?

Back 70

a) Asparagine residue b) Dolichol phosphate c) UDP-N-acetyl glucosamine d) GDP-mannose e) UDP-glucose Occurs mostly in the endoplasmic reticulum

Front 71

The term translocation may occur in 3 places during protein synthesis and processing. What are they and what cofactor does each use?

Back 71

a) Amino acid elongation on the ribosome using EF-G•GDP after peptide bond formation b) Secretion into the ER using Signal Recognition Particle during peptide synthesis. c) Asparagine (N) -linked oligosaccharide synthesis using dolichol phosphate after adding N-acetylglucosamine & mannose residues

Front 72

Where do proteins become targeted to lysosomes?

Back 72

In the Golgi where a phosphate residue is added to a terminal mannose residue on an asparagine-linked glycan on the enzyme

Front 73

What are the similarities between chaperones and chaperonins? Which reacts with a newly synthesized protein first?

Back 73

They are both heat shock proteins that bind unfolded protein along with ATP and release the correctly folded protein when ATP is hydrolyzed

Front 74

What is the function of lysosome-targeted proteins and how does this function differ from proteasomes?

Back 74

Lysosomal proteins hydrolyze extracellular proteins and non-viable cells. Proteasomes remove dysfunctional intracellular proteins within viable cells NOTE: Lysosome-targeted proteins are hydrolytic enzymes, not exclusively proteases.

Front 75

What is a ribosome, and what does it do?

Back 75

is an RNA protein particle on which protein are synthesized

Front 76

What is a proteosome, and what does it do?

Back 76

is a protein particle (26s) that digests ubiquitinylated proteins in the cytosol

Front 77

What is a spliceosome and what does it do?

Back 77

is an snRNA particle that removes introns as eukaryotic mRNA is being synthesized

Front 78

What is a exosome and what does it do?

Back 78

is a protein exonuclease particle that digests all types of RNA from 3' "5'. In eukaryotes, the mRNA cap and tail are removed by endonucleases and the fragments by exonucleases, mostly in exosomes

Front 79

What is a nucleosome, and what does it do?

Back 79

Is a large section of DNA bound to histones and not transcribed unless an activator first removes the histones

Front 80

How many enzymes are used to ubiquinate a protein? And what are their names?

Back 80

Three;Ubiquitin ligases E1, E2 and E3

Front 81

How do these enzymes used to ubiquinate a protein work?

Back 81

E1 activates ubiquitin by attaching its C-terminus to form a thiol ester of cysteine residue on E1. E1 transfers the attached ubiquitin to form a thiol ester on a corresponding cysteine residue on one of a few E2 ligases. Targets are recognized by one of many E3 ligases and they transfer the ubiquitin from the E2 thiol ester to the #-amino group of an internal lysine residue on the target. Lysine residues within the attached ubiquitin then receive additional ubiquitin residues (poly-ubiquitination).

Front 82

Where on a target protein does ubiquitination occur? d) What feature of ubiquitination determines the alternative fates of proteins that have been ubiquinated?

Back 82

The fate of ubiquitinated proteins depends on whether their lysine has received more or less than 4 ubiquitin residues. If less than 4, the target is activated to some function and if more than 4, the target is moved to the proteasome and destroyed.

Front 83

hat enzyme is the initial target of transcription coupled repair?

Back 83

RNA PolII which a DNA mutation has caused to stall during transcript elongation

Front 84

What is the result to the enzyme and to its substrate? What is the mediator of this reaction?

Back 84

Targets enzyme to proteasome and recruits repair enzymes to the DNA substrate and Covalent ubiquitin binding to PolII d) Phosphate residues on the CTD (C-terminal Domain of PolII)

Front 85

Describe three characteristics of a prokaryotic mRNA transcript that differentiates it from a eukaryotic transcript?

Back 85

Prokaryotic mRNA transcripts 1. have no introns
2. are not processed
3. are translated immediately because they are rapidly degraded