Lecture 2: Protein Synthesis and Maturation Flashcards Preview

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Flashcards in Lecture 2: Protein Synthesis and Maturation Deck (59):
1

Definitaion: Gene Expressoin

 

- process by which DNA directs protein synthesis

1. transcription: DNA in gene to RNA

2. translation: RNA sequence to protein

2

Prokaryote Gene expression

 

DNA --> mRNA --> protein

3

Eukaryote gene expression

 

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

 

transcription      processing        translation

4

DNA structure

- 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

 

5

Nucleotide Base Pairs

- Adenine with Thymine

- Guanine with Cytosine

 

A and G are purines (Larger)

C and T are pyrimidines (smaller)

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6

Properties of RNA

 

- 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)

7

Types of RNA

- messenger, mRNA: encodes proteins during translation

- transfer, tRNa: aids translation

- robosomal, rRNA: essential part of ribosomes

8

Process of Transcription (three stages)

 

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

9

What order is the mRNA strand BUILT in transcription?

 

What order is the template strand read?

 

- 5' --> 3'

 

- red in: 3' --> 5'

10

Sense and antisense strands

 

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

Structure of RNA Polymerase

- 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

12

Transcription - Elongation: role of RNA polymerase

 

- 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

13

RNA polymerase vs DNA polymerase

- RNA polymerases do not require a primer to begin transcription

- Lack proofreading function

14

Eukaryotic RNA polymerases

 

- RNA polymerase I

- RNA polymerase II

- RNA polymerase III

 

each transcribes a specific set of genes

15

TRanscription: Promoters of Eukaryotes

- 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

16

Transcription: Bacterial Promoters and binding

σ70 binds to promoter at -10 and -35

this is 40-50 bp away from start site

- 35 box is the consequence sequence TTGACA

17

Transcription - Initiation: Eukaryotes

 

 

-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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Transcription: Initiation for prokaryotes

 

- 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

19

Transcription: Elongation

- 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

20

Transcription: Termination signal in prokaryotes

 

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

21

Transcription: Termination signal in eukaryotes

 

- 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!

22

Special Features of processing Eukaryotic pre-mRNA

 

- add cap at 5' end

- add tail at 3' end

- remove introns and splice exons together

 

- processed mRNA then translocated into cytoplasm

23

Modification after transcription: Importance of RNA splicing?

- 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

 

24

Ways to splice introns

 

- 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

Spliceosome mechanism

 

- 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

Order of snRNP binding

-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

Roles of snRNPs

 

- 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

RNA splicing: Alternative splicing

 

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

Codons

 

- how mRNA is translated

- sequence of 3 nucleotides specifies a particular amino acid

 

 

30

tRNA

 

- acts as a molecular interpreter (or adapter)

- carries amino acids

- matches amino acids with codons in mRNA using anticodons

31

Codon --> amino acid

 

- 64 sense codons to code 20 amino acids

- tRNAs carry complimentary anticodons

32

Translation location: ribosome

 

X

33

Translation: ribosomes structure

 

- 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

start codon

AUG

35

stop codon

 

UAA, UAG, UGA

36

Ribosomal stage of Translation: Iniation

 

- initiation brings together

- start codon ins AUG

- first amino acid is always Met

37

Initiation of Translation: Prokaryotes

 

- mRNA recognition site "upstream" from the start codon

- shine dalgarno sequence

38

in initiation of trasnlation in eukaryotes

 

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

elongation in ribosomal stage of translation (3 steps)

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

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40

Termination of ribosomal translation

 

- 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

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41

Self Splicing introns

 

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

 

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42

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

 

Modified/methylated guanine at end

occurs during elongation

 

- made at phosphorylated c terminus of RNA polymerase II

- 4 enzymatic activities occur

43

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

- 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

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

I: rRNA (ribosomal)

II: protein coding

III: tRNA

45

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

 

- prokaryotic - physical (p independednt/hairpin, p dependent/protein gets in way)

- eukaryotic - release signal

46

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

- alternative splicing allows for multiple results from 1 set of info

the genome is smaller than the transcriptome

47

What is a transriptome?

- human genome is just 25,000 genes

- 8-10x more varaiants produced from splicing

48

Bonding between A-T? Significance?

 

- TWO hydrogen bonds

 

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

49

Bonding between C and G?

 

 THREE hydrogen bonds

50

What are the bonds formed in synthesizing a mRNA strand?

First: hydrigen bonds between complimentary base pairs

Second: phosphodiester linkage of backbone

51

DNA nontamplate: CGCTATAGCGTTT

DNA template:      GCGATATCGCAAA

 

What is the RNA?

 

RNA strand: CGCUAUAGCGUUU

52

Variations of Eukaryotic RNA Polymerase

 

- at least 3 types in eukaryotic organisms

- RNA polymerase I - rRNA genes

- RNA polymerase II - protein coding genes

- RNA polymerase III - tRNA genes

53

Compare the core enzymes of bacterial and eukaryotic RNA polymerases

 

- bacterial have about 5 components

- eukaryotic have about 11-12 (many more!)

54

Importance of phosphorylation of the RNA Polymerase II

 

- 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

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55

Group II vs spliceosome

 

- 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

alternative splicing patterns

 

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57

Alternative splicing of tropomyosin

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58

Prokaryotic vs eukaryotic gene expression

TRanscription and translation occurrence:

gene structure:

modification of mRNA:

 

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

How many codon reading frames are there?

3