Lecture 2: Protein Synthesis and Maturation Flashcards
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)
- Initiation
- RNA polymerase binds to a promotre sequence and forms a transcription bubble
- template strand of DNA is used, other strand is nontemplate - Elongation
- built in 5’ –> 3’ direction - 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
- 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 - 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
Codons
- how mRNA is translated
- sequence of 3 nucleotides specifies a particular amino acid
tRNA
- 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
X
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
AUG
stop codon
UAA, UAG, UGA
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)
- codon recognition: the anticodon of an incoming tRNA pairs with the mRNA codon at the A site
- peptide bond formation: the ribosome catalyzes covalent bond between amino acids at the A and P site
- 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
III: tRNA
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 nontamplate: CGCTATAGCGTTT
DNA template: GCGATATCGCAAA
What is the RNA?
RNA strand: CGCUAUAGCGUUU
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:
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
How many codon reading frames are there?
3
