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Biochem > Lecture 2: Protein Synthesis and Maturation > Flashcards

Lecture 2: Protein Synthesis and Maturation Flashcards

1
Q

Definitaion: Gene Expressoin

A
  • process by which DNA directs protein synthesis
    1. transcription: DNA in gene to RNA
    2. translation: RNA sequence to protein
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2
Q

Prokaryote Gene expression

A

DNA –> mRNA –> protein

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3
Q

Eukaryote gene expression

A

gene/DNA –> primary RNA –> mRNA –> protein

transcription processing translation

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4
Q

DNA structure

A
  • double helix
  • polymer of nucleotides: Adenine, Guanine, Cytosine, Thymine
  • A - T and C - G
  • deoxyribose phosphate backbone
  • antiparallel strands, 5’ phosphate group at one end and 3” hydroxyl group at other end
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5
Q

Nucleotide Base Pairs

A
  • Adenine with Thymine
  • Guanine with Cytosine

A and G are purines (Larger)

C and T are pyrimidines (smaller)

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6
Q

Properties of RNA

A
  • ribose instead of deoxyribose
  • Uracil instead of Thymine (pyrimidine)
  • single stranded but can fold into compact structures with specific functions (tRNA)
  • A-U and C-G
  • synthesized using DNA as a template in transcription
  • some types act as storage (ribosomes) and others as catalysts (ribozymes)
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7
Q

Types of RNA

A
  • messenger, mRNA: encodes proteins during translation
  • transfer, tRNa: aids translation
  • robosomal, rRNA: essential part of ribosomes
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8
Q

Process of Transcription (three stages)

A
  1. Initiation
    - RNA polymerase binds to a promotre sequence and forms a transcription bubble
    - template strand of DNA is used, other strand is nontemplate
  2. Elongation
    - built in 5’ –> 3’ direction
  3. Termination
    - stops at terminator sequence
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9
Q

What order is the mRNA strand BUILT in transcription?

What order is the template strand read?

A
  • 5’ –> 3’
  • red in: 3’ –> 5’
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10
Q

Sense and antisense strands

A

Antisense = template

Sense = compliment to template

Template is read by the mRNA, the mRNA is built to be the same as the non-template strand

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11
Q

Structure of RNA Polymerase

A
  • core enzyme with σ subunit
  • sigma subunit acts as a regulatory fator, guiding the core RNA polymerase to specific promoter sequences on the DNA template strand
  • most have various sigma subunits
  • sigma subunit is what recognizes the TATA box or start sequence

σ70 important for bacteria

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12
Q

Transcription - Elongation: role of RNA polymerase

A
  • DNA has to be unwound near the promoter sequence
  • transcription bubble has to form, sigma subunit has to be released and replaced by NusA
  • RNA polymerase performs a template directed synthesis in a 5’ –> 3’ direction
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13
Q

RNA polymerase vs DNA polymerase

A
  • RNA polymerases do not require a primer to begin transcription
  • Lack proofreading function
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14
Q

Eukaryotic RNA polymerases

A
  • RNA polymerase I
  • RNA polymerase II
  • RNA polymerase III

each transcribes a specific set of genes

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15
Q

TRanscription: Promoters of Eukaryotes

A
  • more diverse and complex series of promoters than prokaryotes
  • TATA box 30 base pairs upstream of transcription start site
  • RNA polymerases do not bund directly to promoter –> a group of proteins called general transcription factors bind to the DNA promoter and RNA polymerase, thus initiating transcription

transcripton factors + RNA polymerase = transcription initiation complex

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16
Q

Transcription: Bacterial Promoters and binding

A

σ70 binds to promoter at -10 and -35

this is 40-50 bp away from start site

  • 35 box is the consequence sequence TTGACA
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17
Q

Transcription - Initiation: Eukaryotes

A
  • promoter: TATA box 30 bp upstream of start site
  • transcription factors bind and mediate
  • RNA polymerase II binds the transcription factors
  • transcription initation complex forms
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18
Q

Transcription: Initiation for prokaryotes

A
  • promoter with sigma subunit recognizes start sequence σ70
  • start sequence in -10 to -35, 20-50 bp with 2 key regions recognized by the sigma subunit
  • 10 sequence is TATAAT sequence to start
  • 35 is consequence sequene
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19
Q

Transcription: Elongation

A
  • DNA unwinds near the promoter
  • transcription bubble forms
  • σ released, replaced by NusA
  • template read 3’ –> 5’
  • built 5’ –> 3’
  • 3’ is nucleophile and attachs alpha phosphate
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20
Q

Transcription: Termination signal in prokaryotes

A
  1. p-independent termination (physical)
    - RNA polymerase encounters a transcription termination signal in the DNA template, coding for RNA that forms a hairpin structure and thus causes RNA polymerase to separate from the RNA transcript
  2. p-dependent termination (physical)
    - p helicases binds on a specific RNA site (rut site) starts to migrate and eventually eparates the RNA from the DNA template
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21
Q

Transcription: Termination signal in eukaryotes

A
  • polyadenylation signal: termination of mRNA synthesis normally occurs when RNA polymerase II has transcribed past a consensus AAUAAA sequence
  • ordinary RNA transcript is cleaved by a special endonuclease 10-35 nucleotides downstream of the signal to generate a new 3’ OH end which is used for further modification (add poly A tail)
  • polyadenylation signal is different from the polyA tail –> it signals where to add the poly A tail!
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22
Q

Special Features of processing Eukaryotic pre-mRNA

A
  • add cap at 5’ end
  • add tail at 3’ end
  • remove introns and splice exons together
  • processed mRNA then translocated into cytoplasm
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23
Q

Modification after transcription: Importance of RNA splicing?

A 
- genes are composed of exons (coding regions)
 and introns (noncoding regions)
  • introns are excised and exons must be linked to form matured mRNA in a post-transcriptiona; process called RNA splicing
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24
Q

Ways to splice introns

A
  • parts of the gene that must be removed
    1. self splicing - splice themselves (I and II)
    2. splicosomal introns - require a large ribonucleoprotein complex (splicosome) to convert pre-mRNA into matures mRNA
    3. ATP + endonuclease (t RNA specifically)
25
Q

Spliceosome mechanism

A
  • spliceosome is a multicomponent complex of small nuclear ribonucleoprotein particles snRNPs consusting of small nuclear RNAs associated with RNA-binding protein
  • formation of the spliceosome and rearrangements of the nucleoproteins within this compelx are preformed by an ATP powered RNA helicase
  • resembles spliceosome action of Group II introns
    1. RNAs bind
    2. make a complex
    3. actively remove introns
26
Q

Order of snRNP binding

A
  • U1 and U2 bind to sepcific intron sites
  • U4, U5, U6 snRNPs then interact with the U1-U2-intron complex to form an inactive spliceosome
  • internal reassembling converts this species to an active spliceosom with U2 ad U6 as its catalytic center, releasing the intron and pslicing the exon ends together
27
Q

Roles of snRNPs

A
  • U1: binds 5’ splice site
  • U2: binds the branch site and forms part of the catalytic center
  • U5: binds the 5’ site and then the 3’ site
  • U4: masks the catalytic activity of U6
  • U6: catalyzes splicing
28
Q

RNA splicing: Alternative splicing

A

purpose: mechanism that generates protein diversity using a limited number of genes
- different combinations of exons from the same gene may be spliced into matured RNA, producing distinct forms of a protein for specific tissues, developmental stages, or signaling pathways
- about 95% of human structural genes are subject to at least one alternative splicing event
- determined by binding of trans-acting splicing factors (activators or repressors) to cis-acting pre-mRNA sequences

29
Q

Codons

A
  • how mRNA is translated
  • sequence of 3 nucleotides specifies a particular amino acid
30
Q

tRNA

A
  • acts as a molecular interpreter (or adapter)
  • carries amino acids
  • matches amino acids with codons in mRNA using anticodons
31
Q

Codon –> amino acid

A
  • 64 sense codons to code 20 amino acids
  • tRNAs carry complimentary anticodons
32
Q

Translation location: ribosome

A

X

33
Q

Translation: ribosomes structure

A
  • 2 protein subunits (large and small) that both contain (catalytically active) ribosomal RNA (rRNA)
  • large subunit: 3 binding sites (APE)

A: amino acid : binds with anticodon of charged tRNA

P: polypeptide: where tRNA adds its amino aid to the growing chain

E: exit: site where tRNA sits before being released fromt he ribosome

mRNA binds between the large and small subunits

34
Q

start codon

A

AUG

35
Q

stop codon

A

UAA, UAG, UGA

36
Q

Ribosomal stage of Translation: Iniation

A
  • initiation brings together
  • start codon ins AUG
  • first amino acid is always Met
37
Q

Initiation of Translation: Prokaryotes

A
  • mRNA recognition site “upstream” from the start codon
  • shine dalgarno sequence
38
Q

in initiation of trasnlation in eukaryotes

A

40s component binds to the 5’ cap on the mRNa and moves until it reaches the start codon

(poly a tail also associates with the 40s subunit)

39
Q

elongation in ribosomal stage of translation (3 steps)

A
  1. codon recognition: the anticodon of an incoming tRNA pairs with the mRNA codon at the A site
  2. peptide bond formation: the ribosome catalyzes covalent bond between amino acids at the A and P site
  3. Translocation: tRNA leaves the P site to the E site of the ribosome, which ejects uncharged tRNA at the E site and moves down
40
Q

Termination of ribosomal translation

A
  • elongation continues until the A site encounters a stop codon, which causes a socalled “Release factor” to enter the site
  • release factor RESEMBLES tRNA in size and shape but does not carry an amino acid
  • allows the hydrolysis of the bond linking the tRNA in the P site with the polypeptide chain
  • polypeptide chain dissociates from the ribosome and folds into its active conformation
41
Q

Self Splicing introns

A

Group I:

  • needs GDP
  • cuts the sequence on 5’ side
  • 5’ side attacks 3’ end
  • guanine nucleoside important

Group II:

  • ithin intron a lariat forms
  • makes a cut ont he 5’ side
  • 5’ nucleophile attacks the 3’ side

* only possible if intron is not too long

42
Q

What is the 5’ Cap? When is it made? Where?

A

Modified/methylated guanine at end

occurs during elongation

  • made at phosphorylated c terminus of RNA polymerase II
  • 4 enzymatic activities occur
43
Q

What is the 3’ poly A tail? where is it made? when?

A
  • 30-250 A residues
  • transcript added beyond site, cleaved at AAA site
  • 3’ OH added at the end
  • made at c terminus of RNA polymerase II
  • end of transcription
44
Q

What are the main types of RNA polymerases and their uses?

A

I: rRNA (ribosomal)

II: protein coding

III: tRNA

45
Q

Transcription: what is the main difference between prokaryotic and eukaryotic termination?

A
  • prokaryotic - physical (p independednt/hairpin, p dependent/protein gets in way)
  • eukaryotic - release signal
46
Q

Why do humans have the more “complicated” flow of genetic information that includes RNA processing?

A
  • alternative splicing allows for multiple results from 1 set of info

the genome is smaller than the transcriptome

47
Q

What is a transriptome?

A
  • human genome is just 25,000 genes
  • 8-10x more varaiants produced from splicing
48
Q

Bonding between A-T? Significance?

A
  • TWO hydrogen bonds

TATA box is a section with many of these –> easier to break so signals start of transcription

49
Q

Bonding between C and G?

A

THREE hydrogen bonds

50
Q

What are the bonds formed in synthesizing a mRNA strand?

A

First: hydrigen bonds between complimentary base pairs

Second: phosphodiester linkage of backbone

51
Q

DNA nontamplate: CGCTATAGCGTTT

DNA template: GCGATATCGCAAA

What is the RNA?

A

RNA strand: CGCUAUAGCGUUU

52
Q

Variations of Eukaryotic RNA Polymerase

A
  • at least 3 types in eukaryotic organisms
  • RNA polymerase I - rRNA genes
  • RNA polymerase II - protein coding genes
  • RNA polymerase III - tRNA genes
53
Q

Compare the core enzymes of bacterial and eukaryotic RNA polymerases

A
  • bacterial have about 5 components
  • eukaryotic have about 11-12 (many more!)
54
Q

Importance of phosphorylation of the RNA Polymerase II

A
  • coupling of pre-mRNA transcription and processing –> it is the site of tha processing
  • c terminal part
  • contains all important enzymes
  • the phosphorylated c terminus is involved in doing most activities such as adding the poly a tail, 5’ cap, spliceosome
  • everything is done within the proximity fo the RNA complex
55
Q

Group II vs spliceosome

A
  • group II is possible when the intron is not too long
  • if the intron is too long a spliceosome is needed
  • similar in that they form lariat structures
56
Q

alternative splicing patterns

A
57
Q

Alternative splicing of tropomyosin

A
58
Q

Prokaryotic vs eukaryotic gene expression

TRanscription and translation occurrence:

gene structure:

modification of mRNA:

A

Prokaryote:

  • at same time in cytoplasm
  • DNA read in same order as amino acid
  • no modification

Eukaryote:

  • transcription in nucleus then translation in cytoplasm
  • noncoding introns within coding sequence
  • introns spliced, 5’cap, poly A tail
59
Q

How many codon reading frames are there?

A

3

Biochem (15 decks)

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