Translation (Protein Synthesis)

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PROTEIN SYNTHESIS

Translation

Conversion of mRNA to protein

Codon

Group of three mRNA nucleotides that encode one amino acid

Ribosomal Structure

  • 2 subunits each made of protein and RNA:
  1. Small ribosomal subunit – has mRNA binding site
  2. Large ribosomal subunit
  • E-site (Exit site)
  • P-site (Peptidyl-tRNA binding site)
  • A-site (Aminoacyl-tRNA binding site)

PROTEIN SYNTHESIS STEPS

Initiation

  • Initiator t-RNA (methionine) binds to small ribosomal subunit (with help from initiation factors)
  • Small ribosomal subunit/initiator t-RNA complex binds mRNA (using 5′ cap to bind the 5′ end)
  • Complex scans towards the 3′ end until it locates the start codon (AUG which sets the appropriate reading frame) and the large ribosomal subunit arrives

Elongation

  • Current tRNA in the P-site
  • New tRNA added to A-site
  • Growing peptide chain added to amino acid bound to A-site tRNA
  • Large ribosomal subunit shifts 3′ one codon
  • Small ribosomal subunit follows – tRNA that was in P-site now in E-site and tRNA that was in A-site now in P-site
  • E-site tRNA ejected and new A-site tRNA added

Termination

  • When codon in A-site is stop codon (UAG, UAA, or UGA) a release factor is added to the A-site
  • Release factor binding releases the peptide chain and causes ribosome to dissociate

Transcription, Part II

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POST-TRANSCRIPTIONAL MODIFICATIONS:

  • Help with the export of the mRNA from the nucleus
  • Protect the mRNA from degradation in the cytoplasm
  • Help facilitate proper translation

3 TYPES OF MODIFICATIONS

  • 5′ capping
  • Poly-A tail
  • Intron splicing

5′ capping

  • Modified guanine nucleotide
  • Added to 5′ end of RNA molecule
  • Added during transcription

Poly-A tail

  • Multiple adenines added at the end of the polyadenylation signal
  • Added to the 3′ end of the RNA

Intron splicing

Intron

Sections of the RNA sequence that are found in between exons

  • Introns are removed from the RNA by the spliceosome

Spliceosome

Group of small nuclear RNAs and proteins which join together to recognize and remove introns from transcribed RNA

  • Products of intron splice are mature mRNA and circular intron

Alternative splicing allows for one gene to code for multiple, similar proteins

Incorrect splicing is often the cause of the disease Hutchinson-Gilford progeria syndrome

DIFFERENCES BETWEEN PROKARYOTIC AND EUKARYOTIC TRANSCRIPTION

Prokaryotes:

  • Only one polymerase isoform
  • Occurs in the cytoplasm (since no nucleus)
  • RNA polymerase recognizes the promoter sequence
  • RNA polymerase recognizes the terminator sequence
  • No additional processing of mRNA

Transcription, Part I

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CONVERTS INFORMATION IN DNA TO RNA

  • Protects original information
  • Multiple RNA copies allow for more protein to be produced

DEFINITIONS

Transcription factors

Proteins which bind the promoter and recruit the RNA polymerase

Template strand

Strand of DNA in which the gene to be transcribed is located

THREE STEP PROCESS:

Initiation

  • Promoter recognized by transcription factors
  • RNA Polymerase II recruited by the transcription factors
  • Transcription begins and continues 3′ to 5′ along template strand

Elongation

  • Complementary RNA nucleotides added to the growing RNA strand (uracil instead of thymine in the RNA strand)

Termination

  • Polyadenylation signal in the transcribed RNA signals proteins to cut it from the polymerase
  • Newly formed RNA is called Pre-mRNA because it still needs to be processed
  • RNA Polymerase eventually falls off the template strand

Gene Expression Regulation Overview

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  • Allows organisms to be more efficient with resources and to adapt to the environment
  • Important for cell differentiation (which results in many different cell types)

STEPS REGULATION OCCURS

  1. Transcriptional Control
  • When and how often a gene is transcribed
  • Only point where the cell will not synthesize unneeded intermediates, thereby wasting resources
  1. RNA Processing Control
  • Control of the splicing and processing of RNA transcripts
  1. RNA Transport and Localization Control
  • Cells select which mRNAs are exported to the cytoplasm and where they are localized to
  1. Translational Control
  • Control which mRNAs get translated into protein
  1. mRNA Degradation Control
  • Cells can destabilize specific mRNAs in the cytoplasm to limit protein production
  1. Protein Activity Control
  • Cells can selectively activate, inactivate, localize or degrade proteins after they have been synthesized

Excision Repair

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EXCISION REPAIR MECHANISMS

Mismatch-repair

• Fixes replication errors missed by DNA Pol proofreading(cannot repair damage)

Base excision repair

• Damaged bases removed and replaced
• Deamination & depurination (most common spontaneous damages)
• Alkylation (except for O6-alkyl guanine)
• Oxidation

Nucleotide excision repair

• Damaged nucleotides removed and replaced
• 2 types: global genomic NER (transcriptionally inactive) & transcription-coupled
• Repairs wider variety of lesions that distort DNA helix
• Pyrimidine dimers and other intrastrand adducts
• Bulky group addition (via carcinogen-exposure)

BASE EXCISION REPAIR
• Model: cytosine spontaneously deaminates to uracil

  1. DNA glycosylase excises uracil: cleaves N-glycosidic bond between base & deoxyribose
    • Produces apyrimidinic site (AP site)
  2. AP endonuclease cleaves deoxyribose backbone adjacent to AP site
    • Excises deoxyribose moiety
  3. DNA polymerase and ligase fill/seal gap with cytosine
    • Prokaryotes: DNA Pol. I
    • Eukaryotes: DNA Pol. beta

NUCLEOTIDE EXCISION REPAIR
• Model: thymine dimer in E. Coli (prokaryotes)

Global genomic NER: transcriptionally inactive region

• Uvr genes: discovered in E. Coli, mutations conferred extreme UV light sensitivity

  1. UvrA recognizes helical distortion as damage
  2. UvrA recruits uvrB and uvrC

Intertextual variation exists for above step.

  1. Uvr B and C cleave DNA on either side of damage
    • Uvr ABC complex: excinuclease
  2. Helicase unwinds DNA and releases damaged segment
  3. DNA polymerase fills gap and ligase seals it
    • DNA Pol. fills gap in 5′ to 3′ direction
    • Prokaryotes: DNA Pol. I
    • Eukaryotes: DNA Pol. delta and epsilon

Transcription-coupled NER: transcriptionally active region

• Repairs more rapidly than global genomic pathway

  1. NER enzymes recognize RNA polymerase stalled by lesion and displace it
    • E. Coli: NER enzymes are Mfd proteins
    • Humans: RNA polymerase II (prokaryotes only have one RNA Pol.) & CS proteins
  2. Mfd proteins (E. Coli) and CS proteins (humans) recruit other proteins to site
    • E. Coli: Mfd proteins recruit uvr proteins
    • Humans: CS proteins recruit more CS and XP proteins (amongst others)
  3. Recruited proteins cleave and excise damaged oligomer
  4. Identical to Step 5 in global genomic pathway

CLINICAL CORRELATION

Xeroderma pigmentosum (XP)

• Rare genetic defect produces dysfunctional XP proteins
• Patients extremely sensitive to UV light: develop skin cancers in sun-exposed areas

Mismatch Repair

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DNA REPLICATION: FACTORS THAT INCREASE FIDELITY
• Increase fidelity to 1 mistake per 10^9 bases added

  1. DNA Pol. proofreading capacity
    • DNA Pol. 3′ to 5′ exonuclease activity: can excise/replace incorrect nucleotides
    • Prokaryotes: DNA Pol. I (replaces RNA primers) & III (daughter strands) are exonucleases
    • Eukaryotes: DNA Pol. delta (lagging strand) & epsilon (leading strand) are exonucleases
  2. Repair mechanisms

Mismatch repair: identifies/fixes replication errors that escape DNA Pol.

• Addresses single-strand breaks in newly replicated DNA
• Cannot repair DNA Damage

MISMATCH REPAIR: E. COLI
• Mismatched nucleotides cannot H-bond –> distorts DNA
• GATC sequence occurs about every thousand nucleotides
• A in GATC is methylated: distinguishes parent strand from daughter strand
• Mut proteins (mismatch repair enzymes in E. Coli)

  1. MutS recognizes mismatched base and initiates repair by binding
  2. MutS forms complex with MutL
  3. MutL binding activates endonuclease MutH
  4. MutH cleaves daughter strand opposite to adenine-methylation (GATC)
  5. Exonuclease and helicase excise portion of daughter strand

Intertextual variation exits regarding above step.

  1. DNA Pol. (III or I) fills gap and ligase seals ends w/ phosphodiester bonds
    • DNA Pol synthesizes gap in 5′ to 3′ direction

MISMATCH REPAIR: HUMANS
• MSH proteins: human homologs for Mut proteins (no MutH homolog)
• Daughter strand specificity poorly understood: not adenine-methylation

Theoretical recognition sites: breaks in lagging strand & lengthening 3′ end of lagging strand.

• DNA Pol. (delta and epsilon) fill excised gap

CLINICAL CORRELATION

Lynch Syndrome

• Formerly known as hereditary nonpolyposis colorectal cancer (HNPCC)
• Mutation in human Mut homologs: defective mismatch repair pathway
• Increased risk for colorectal and other cancers
• ~ 3 in every 100 cases of colon cancer caused by Lynch syndrome