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Integrated - Block 1 Exam 1 > BCH - Samuelson 8/13 lec part 2 > Flashcards

BCH - Samuelson 8/13 lec part 2 Flashcards

1
Q

Coding RNA is an intermediate in

A

information metabolism

DNA –> mRNA –> protein

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2
Q

Coding RNA?

A

mRNA (messenger)

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3
Q

Noncoding RNA (2 examples)

A

rRNA (ribosomal)
tRNA (transfer)

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4
Q

RNA structure

A

RNA has 2’ hydroxyl group, U in RNA instead of T in DNA, A in DNA is transcribed to U in RNA

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5
Q

RNA synthesis - Transcription

A
  1. reaction catalyzed by DNA dependent RNA polymerases
  2. RNA polymerases enzymes synthesize RNA by adding complementary NTPs to the 3’ OH of the growing strand
  3. RNA polymerases do not edit RNA. No proofreading, no repair
  4. No primer required to start synthesis
  5. Promoter in gene dictates site at which RNA sythesis starts
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6
Q

Explain RNA as the sense strand

A

RNA is a complement of the DNA template strand and RNA version of the non-template (coding) DNA strand

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7
Q

3 steps of RNA synthesis - transcription

A

initiation, elongation, termination

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8
Q

RNA synthesized by RNA polymerases - bacteria

A

Single RNA polymerase

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9
Q

RNA synthesized by RNA polymerases - eukaryotes

A

3 RNA polymerases

  1. RNA polymerase 2: mRNA, snRNAs, miRNAs

RNA polymerase 2 synthesizes mRNA

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10
Q

What is the start site of transcription controlled by

A

the gene promoter

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11
Q

What is the transcription start site

A

Pribnow box

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12
Q

Enzymes use what to identify the Pribnow box (transcription start site)

A

AT rich regions

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13
Q

where is the Pribnow box located

A

5’ of and approximately 10 bases from the transcription start site

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14
Q

What is the pribnow box important for

A

for recruiting RNA polymerase to this spot

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15
Q

RNAP sigma factor has a major function in

A

transcription initiation

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16
Q

How does sigma factor work

A

RNA pol holo enzyme with sigma binds to promoter sequences with tight binding

Conversely, RNA pol holo enzyme binds to non-promoter DNA sequences with loose binding

core RNA polymerase without sigma factor binds promoter or non-promoter DNA with equal affinities

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17
Q

What does Kd stand for

A

dissociation constant

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18
Q

Bacterial RNA polymerase - after promoter recognition, what happens with sigma?

A

sigma dissociates from the holoenzyme leaving the core enzyme to complete RNA synthesis

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19
Q

RNA elongation follows initiation - RNA synthesis is

A

processive in a 5’ to 3’ direction at a rate of ~20-50 nt/second

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20
Q

Bacterial transcription initiation

A

RNA polymerase and bound sigma factor scan along DNA.

Sigma factor required to identify the transcription start
site and initiate transcription.

  • Polymerase separates ~11 bp of DNA to allow RNA
    synthesis.
  • Different sigma factors recognize different
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21
Q

Bacterial Transcription - Elongation

A

After the first phosphodiester bond is formed, the sigma factor dissociates, and RNA is
synthesized

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22
Q

RNA pol 2 is required for

A

synthesis of mRNAs

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23
Q

Transcription initiation

A

most genes (50-60%) have an upstream TATA box element at -30 to -100

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24
Q

Talk about eukaryotic gene promoters of transcription (initiation)

A

Tightly wound DNA needs to be opened

Transcription factors begin to open this up

TATA box is where basal transcription factors set up 1st and they recruit RNA polymerase 2

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25
Transcription - eukaryotes
TATA binding protein binds, following by basal transcription factors, which are common to all genes transcribed by pol 2 Once the complex is assembled, RNAP 2 is phosphorylated and elongation begins
26
RNA processing
Primary (nascent) transcript not usually active: requires processing - nucleolytic cleavage - terminal additions - base and nucleotides modifications
27
mRNA processing - synthesized by RNA polymerase 2
1. Pre-mRNAs are large precursors with introns (eukaryotes) Processing requires 3 steps for most mRNAs 1. additions of 5' CAP - 7MeG linked by triphosphate linkage 2. addition of polyA tail (200 A's added to 3' end) 3. Removal of introns (RNA splicing)
28
5' CAP on mRNAs 5'-methyl guanosine cap linkage is
5'-5' and triphosphate
29
RNA splicing
removing introns and joining exons to make mature mRNA site specific cleavage and ligation spliceosome is structure including snRNAs (small nuclear RNAs), U1 and U2 snRNA
30
PolyA addition to mRNA
poly A added at consensus sequence (AAUAAA)
31
RNA editing - another modification of mRNA
RNA editing in human intestine there is a specific cytosine deaminase that binds to the mRNA and dominates a specific C. This results in C to U and change of a codon from CAA (Gln) to UAA (stop) in specific mRNAs after transcription, protein can edit RNA to make different products
32
Alternative splicing increases
diversity can make things more complex because different gene products from same gene differently spliced = different mRNAs
33
Explain the genetic code
it is used to translate information stored in DNA into proteins, is evolutionarily conserved
34
Translation - Protein Synthesis *Reading Frames*
mRNA codes for proteins using common triplets of bases that are recognized by tRNAs. This means every mRNA has 3 potential reading frames. Each reading frame produces a different protein.
35
Translation - Protein Synthesis *Open Reading Frame(ORF)*
First amino acid is always methionine (met) and continues until a stop codon is reached
36
Polypeptide chains are assembled linearly from
the amino terminus to the carboxyl terminus
37
What happens to an amino acid sequence if one nucleotide is added to or removed from the ORF?
A frameshift variant occurs when there is an addition or loss of nucleotides that shifts the grouping and changes the code for all downstream amino acids.
38
Genetic Code - Triple Code
non-overlapping, non-punctuated, triplet codons
39
Genetic Code - Degenerate characteristics
- 20 amino acids - 32 tRNAs - 64 triplet codons (61 code from amino acids, 3 code for termination)
40
Genetic Code - Degenerate definition
multiple codons result in the addition of the same amino acid so a single tRNA must be capable of recognizing multiple codons
41
tRNAs base-pair with mRNA by means of a sequence on the tRNA called the
Anticodon
42
Wobble base pairing
guanine-uracil (G-U), hypoxanthine-uracil (I-U), hypoxanthine-adenine (I-A), and hypoxanthine-cytosine (I-C)
43
tRNA processing
cleavage at 3' end of RNAse D CCA is added to 3' end by terminal transferase
44
tRNA base modification important for tRNA function
Inosine
45
5 steps of protein synthesis
1. activation of amino acids Translation 2. initiation 3. elongation 4. termination Protein processing 5. folding and processing
46
Aminoacyl tRNA syntheses are high fidelity enzymes - catalytic site is a dinucleotide fold that forms a
pocket that binds nucleosides, ATP, and amino acid
47
Aminoacyl tRNA syntheses are high fidelity enzymes - an incorrect amino acid -
tRNA pairing is hydrolyzed at the editing site which is a different pocket
48
Amino acid addition to tRNA - catalyzed by
aminoacyl-tRNA synthetase enzymes separate enzyme for each of the 20 amino acids
49
Amino acid addition to tRNA - 2 step reaction catalyzed by same enzyme
Addition of the amino acid, through its COOH group to the alpha phosphate of ATP Transfer of the amino acid from the aminoacyl adenylate to the 3' or 2' OH of the adenosine on the CCA at the 3' end of the cognate tRNA
50
Activation of amino acids - Summary
2 step reaction: Overall reaction: Amino acid + tRNA +ATP -> aminoacyl-tRNA +AMP + 2Pi a. one enzyme per amino acid b. high substrate specificity and proofreading (can remove amino acid if incorrect aa was added) = high fidelity c. amino acid attached to 2’ or 3’ OH of the 3’ terminal A on the tRNA. d. ability of aminoacyl tRNA synthetases to recognize amino acids and cognate tRNAs is critical. This interaction is referred to as the second genetic code
51
Protein synthesis - translation
a regulated RNA-dependent biochemical process that results in the synthesis of amino acid sequences or polypeptides
52
Protein synthesis on ribosome
Process is very similar in prokaryotes and eukaryotes, but details differ, and this is important for human health Many antibiotics target bacterial translation. Specificity of antibiotics is because of these differences
53
Shine-Dalgarno sequence
upstream of AUG Ribosomal subunit binds to Shine-Dalgarno sequence then scans to the first AUG and positions this codon over the P site (peptidyl)
54
Translation - Initiation
Assembly of mRNA, Ribosome and fMet-tRNAMet a. ribosomal subunits b. mRNA c. initiation factors (IF) d. fMet-tRNAfMet (bacteria) 1. Two initiation factors (IF-1 and IF-3) bind the small (30S) ribosomal subunit (IF-3 stops large ribosomal subunit binding prematurely) 2. The mRNA binds to the small ribosomal subunit. Initiating codon (AUG) is guided to correct location by Shine-Dalgarno sequence, upstream of AUG. Ribosomal subunit binds to Shine-Dalgarno sequence then scans to the first AUG and positions this codon over the P site (peptide) 3. fMet-tRNAf Met and IF-2 with GTP bound, bind to the complex. 4. GTP hydrolyzed and IF depart, and large subunit (50S) binds to complex
55
Translation Initiation in Bacteria
contains an amide bond; therefore, fMet can only be the N-terminal residue
56
Elongation
Elongation: stepwise addition of amino acids to the peptide chain. A. Requirements (bacteria) a. initiation complex b. elongation factors (EF-Tu, EF-Ts, EF-G) c. charged tRNAs (aminoacyl tRNAs) d. GTP STEPS: 1. Position next charged tRNA at A site. EF-Tu with bound GTP positions tRNA at the A site, then EF dissociates
57
Elongation - Peptide bond formation
Catalyzed by peptidyl transferase, an activity of the 28S rRNA (acting as a ribozyme)
58
Ribosomal peptidyl transferase reaction
Nucleophilic reaction amino acid group in the A site and the ester in the P site
59
Elongation - Translocation - definition?
Movement of the ribosome with respect to the mRNA – index by three nucleotides (one codon) – place the newly-formed peptidyl tRNA in the P site and provides a vacant A site for entry of the next aminoacyl-tRNA. – The empty tRNA aligned in E (exit) site, and leaves
60
Synthesis of a polypeptide is from the
amino (N) terminus to carboxy (C) terminus translation of the mRNA is from its 5' to 3' end
61
Termination Requirements
in frame stop codon in mRNA (UAA, UAG, UGA) release factors (RFs) bind at A site because there are no tRNAs to out compete for a stop codon
62
Termination Steps
When termination codon arrives to A site, release factors bind to this site and hydrolyze the terminal peptidyl-tRNA bond, releasing the protein. last deacylated tRNA released ribosomal subunits dissociate.
63
Termination - Expensive
4 ATP or GTP per amino acid added, plus one for initiation and termination
64
To increase the speed of translation, each mRNA is
translated into protein by multiple ribosomes. Each mRNA has several ribosomes attached, called polyribosome or polysomes
65
Inhibitors of Translation - Cycloheximide (CHX)
Binds to the E site of ribosomes and inhibits ribosome translocation by blocking the movement of the tRNA from the aminoacyl (A) acceptor site to the peptidyl (P) donor site, thus inhibiting elongation
66

Integrated - Block 1 Exam 1 (5 decks)

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