RNA and the Genetic Code

• Gene = unit of DNA that encodes stuff

Types of RNA

Messenger RNA (mRNA)

• Carries info specifying amino acid sequence
• Made from template DNA by RNA polymerase
• Only RNA that gets translated
• Eukaryotes: mRNA is monocistronic (different mRNA for different proteins)
• Prokaryotes: mRNA is polycistronic (starts translation at different locations)

tRNA

• Converts nucleic acid info → amino acids/peptides
• Folded RNA strand with anticodon
• Charged with amino acid

Aminoacyl-tRNA synthetase

• Requires 2 high-energy bonds from ATP
• Transfers activated amino acid to tRNA

Ribosomal RNA (rRNA)

• Synthesized in nucleolus
• Functions as ribosomal machinery
• Most rRNA are ribozymes
  • Enzymes made of RNA instead of peptides
  • Catalyze peptide bond formation

Codons

• 3-letter word
• $64$ codons
• Recognized by complementary anticodon

Mutations

• Degeneracy: more than one codon can specify a single amino acid
• Wobble: third base can differ (protects against mutations)
• Missense = substitution
• Nonsense = stop codon
• Frameshift = everything messed up

• Transcription = mRNA made from DNA

Mechanism

• Helicase + topoisomerase unwind DNA
• Template strand = antisense strand
• New RNA strand is antiparallel + complementary to template DNA

Promoter region

• RNA polymerase searches promoter regions
• RNA polymerase II is main enzyme transcribing mRNA
• Key promoter site: TATA box (high T/A content)
• Transcription factors help RNA pol bind + locate promoter

RNA polymerase types

• RNA pol I:
  • Nucleolus
  • rRNA
• RNA pol II:
  • Nucleus
  • hnRNA (preprocessed) and snRNA (small nuclear RNA)
• RNA pol III:
  • Nucleus
  • tRNA and some rRNA

Coding strand

• Not used as template
• Complementary to template strand
• Identical to mRNA except T → U

Gene numbering

• First RNA base transcribed = +1 of gene region
• Bases left of +1 are negative, right are positive
• No nucleotide is 0
• RNA pol II binds TATA box around -25

Post-transcriptional processing

• hnRNA must be processed before leaving nucleus for translation

Splicing

• Done by spliceosome
• snRNA couples with snRNPs
• snRNP recognizes both 5’ and 3’ splice sites
• Noncoding sequences removed as lariat (lasso) → degraded

5’ cap

• Protects mRNA from degradation in cytoplasm

3’ poly-A tail

• Protects against rapid degradation
• Acts like a time bomb: mRNA starts degrading after it leaves nucleus
• Longer tail → survives longer
• Mature mRNA transported into cytoplasm
• Alternative splicing → more proteins from limited genes

• mRNA created + processed → exits via nuclear pores → translated

Ribosome

• Made of proteins + rRNA
• Has A, P, E sites
• Eukaryotic ribosomes: 28S, 18S, 5.8S, 5S rRNA
  • RNA pol I transcribes 28S, 18S, 5.8S together as 45S precursor
  • 45S processed → 18S (40S subunit) + 28S + 5.8S (60S subunit)
  • RNA pol III transcribes 5S rRNA (in 60S)
• Subunits formed (40S + 60S) → assemble into 80S ribosome

Mechanism of translation

Initiation

• Small subunit binds Shine-Dalgarno sequence
• Charged initiator tRNA binds AUG start codon
• fMet is first amino acid
• Large subunit binds → initiation complex
• Assisted by initiation factors (IF)

Elongation

• Repeating 3-step cycle for each amino acid
• A site:
  • Incoming aminoacyl-tRNA (next AA added)
• P site:
  • Holds tRNA carrying growing polypeptide chain
  • Where first AA binds
• Requires peptidyl transferase:
  • Catalyzes peptide bond between AA in A site and growing chain in P site
  • Peptide bond = amide bond
• E site:
  • Uncharged tRNA pauses before exiting
• All 3 stages require energy
• Elongation factors (EF) recruit/locate aminoacyl-tRNA using GTP
• Signal sequence can direct ribosome to ER → protein to Golgi → secreted via vesicle

Termination

• When stop codon enters A site, release factor (RF) binds
• Water added to polypeptide chain
• Peptidyl transferase + termination factors hydrolyze chain
• Polypeptide released from tRNA in P site
• Ribosomal subunits dissociate

Post-translational processing

• Protein must fold correctly
• Chaperone proteins assist folding
• Cleavage modifications:
  • Insulin cleaved from larger inactive peptide to active peptide
• Quaternary proteins require subunits to assemble
• Other modifications:
  • Phosphorylation
  • Carboxylation
  • Glycosylation
  • Prenylation

Operon

• On/off switch where genes share common promoter
• Transcribed as a group
• Examples: trp operon, lac operon
• Jacob–Monod model describes operon structure/function
• Operons contain:
  • Structural genes
  • Operator site
  • Promoter site
  • Regulator gene

Structural gene

• Codes for protein of interest

Operator site

• Nontranscribable region
• Binds repressor

Promoter site

• Where RNA polymerase binds

Regulator gene

• Upstream
• Codes for repressor

Inducible systems

• Repressor tightly bound to operator → roadblock
• Negative control mechanism
• Inducer binds repressor:
  • Competitive inhibition idea
  • Higher $$[inducer]$$ pulls more repressors off operator
• Lac operon is inducible
• CAP + cAMP binding enables CAP to bind promoter region
  • Positive control mechanism

Repressible system

• Constant production of protein product
• Repressor inactive until binds corepressor
• Corepressor activates repressor → binds operator → stops transcription
• Trp operon is repressible
  • High tryptophan = corepressor
  • 2 molecules cause repressor to bind operator

Transcription factors (TFs)

• Transcription-activating proteins that search DNA for binding motifs

DNA-binding domain

• Binds specific sequences in promoter or DNA response element
• Recruits transcription machinery

Activation domain

• Binds multiple TFs + regulatory proteins (RNA pol + histone acetylases)
• Helps remodel chromatin

Gene amplification

• Enhancers increase RNA pol ability at a single promoter site
• Group of response elements controls expression of one gene
  • cAMP binds CREB
  • Cortisol binds cortisol receptor
• TFs bind their response elements
• Enhancers can be ~1000 bp away from the gene

Gene duplication

• Cells can duplicate relevant gene → higher expression
• Can also duplicate in parallel by opening genes with helicase
  • Allows replication of only that gene
• Hundreds of copies can exist in parallel

Regulation of chromatin structure

• Heterochromatin:
  • Tightly coiled DNA
  • Dark under microscope
• Euchromatin:
  • Looser DNA
• Chromatin remodeling regulates gene expression

Histone acetylation

• Recruits coactivators like histone acetylases
• Acetylates lysine residues on histone tails
• Acetylation decreases + charge → weakens histone–DNA interaction
• Easier access for transcription machinery to RNA
• Histone deacetylases remove acetyl groups → decrease gene expression

DNA methylation

• Adds methyl groups to cytosine and adenine nucleotides
• Silences gene expression
• Important in development
• Heterochromatin more heavily methylated