Lecture 2: Protein Synthesis and Maturation Flashcards Preview

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

Definitaion: Gene Expressoin


- process by which DNA directs protein synthesis

1. transcription: DNA in gene to RNA

2. translation: RNA sequence to protein


Prokaryote Gene expression


DNA --> mRNA --> protein


Eukaryote gene expression


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


transcription      processing        translation


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



Nucleotide Base Pairs

- Adenine with Thymine

- Guanine with Cytosine


A and G are purines (Larger)

C and T are pyrimidines (smaller)


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)


Types of RNA

- messenger, mRNA: encodes proteins during translation

- transfer, tRNa: aids translation

- robosomal, rRNA: essential part of ribosomes


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


What order is the mRNA strand BUILT in transcription?


What order is the template strand read?


- 5' --> 3'


- red in: 3' --> 5'


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


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


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


RNA polymerase vs DNA polymerase

- RNA polymerases do not require a primer to begin transcription

- Lack proofreading function


Eukaryotic RNA polymerases


- RNA polymerase I

- RNA polymerase II

- RNA polymerase III


each transcribes a specific set of genes


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


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


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




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


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


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


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!


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


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



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)


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


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


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


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




- how mRNA is translated

- sequence of 3 nucleotides specifies a particular amino acid






- acts as a molecular interpreter (or adapter)

- carries amino acids

- matches amino acids with codons in mRNA using anticodons


Codon --> amino acid


- 64 sense codons to code 20 amino acids

- tRNAs carry complimentary anticodons


Translation location: ribosome




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


start codon



stop codon




Ribosomal stage of Translation: Iniation


- initiation brings together

- start codon ins AUG

- first amino acid is always Met


Initiation of Translation: Prokaryotes


- mRNA recognition site "upstream" from the start codon

- shine dalgarno sequence


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)


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


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


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



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


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


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

I: rRNA (ribosomal)

II: protein coding



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


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


What is a transriptome?

- human genome is just 25,000 genes

- 8-10x more varaiants produced from splicing


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


Bonding between C and G?


 THREE hydrogen bonds


What are the bonds formed in synthesizing a mRNA strand?

First: hydrigen bonds between complimentary base pairs

Second: phosphodiester linkage of backbone



DNA template:      GCGATATCGCAAA


What is the RNA?




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


Compare the core enzymes of bacterial and eukaryotic RNA polymerases


- bacterial have about 5 components

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


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


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


alternative splicing patterns



Alternative splicing of tropomyosin


Prokaryotic vs eukaryotic gene expression

TRanscription and translation occurrence:

gene structure:

modification of mRNA:



- at same time in cytoplasm

- DNA read in same order as amino acid

- no modification


- transcription in nucleus then translation in cytoplasm

- noncoding introns within coding sequence

- introns spliced, 5'cap, poly A tail


How many codon reading frames are there?