genetics chapter 14

translation

Biological polymerization of amino acids into peptide chains

translation requires

• amino acids

• mrna

• ribosomes

• trna

ribosomes

• Have an essential role in expression of genetic information

• Consist of ribosomal proteins and ribosomal RNAs (rRNAs)

• rRNAs perform the central catalytic functions associated with translation

rDNA (ribosomal dna)

• rRNA genes

• Moderately repetitive DNA fraction present in clusters at various chromosomal sites

• Each cluster contains tandem repeats (meaning adjacent) separated by noncoding spacer DNA

how are rDNA organized

rRNA genes are organized in clusters (45S) and those clusters are repeated 200 – 400 times on a chromosome

why so many copies?

Cell must make lots of ribosomal RNA to maintain its stock of ribosomes for protein synthesis

trnas adaptor molecule

• Adapt genetic information present as specific triplet codons in mRNA to corresponding amino acid

• tRNA anticodons complement mRNAs

• tRNAs carry corresponding amino acids

trnas

• small and stable

• 75-90 nucleotides

• transcribed from dna as larger precursors, then cleaved into mature tRNA

• contained prosttranscriptionally modified bases, created after tRNA transcription

Bases can confer structural stability, important for hydrogen bonding between tRNA and mRNA

• tRNAs have a cloverleaf structure

anticodon

• tRNA has anticodon that complementarily base-pairs with codon in mRNA

• Corresponding amino acid is covalently linked to CCA sequence at 3’ end of all tRNAs

• Linking the appropriate amino acid to the tRNA is called “charging” the tRNA

Aminoacylation: tRNA charging

Before translation can proceed, tRNA molecules must be chemically linked to respective amino acids

Aminoacyl tRNA synthetase

Enzyme that catalyzes aminoacylation

Aminoacylation is Highly specific:

• The specificity between an AA and its tRNA is determined by each aminoacyl-tRNA synthetase recognize only one amino acid

• 20 different synthetases classes, one for each amino acid

• tRNAs that correspond to that AA are called isoaccepting tRNAs

Aminoacylation: tRNA charging

• AA is converted to an activated form through a reaction with ATP, that covalently links the AMP to the AA

• Aminoacyl tRNA synthetase then transfers the AA to the appropriate tRNA creating a charged tRNA

• Charged tRNA is also referred to as aminoacyl tRNA and it may then participate directly in protein synthesis

initiation requires

• Small and large ribosomal subunits

• mRNA molecule

• GTP

• Charged initiator tRNA

• Mg2+

• Initiation factors

Ribosomes contain 3 sites

• Aminoacyl (A) site

• Peptidyl (P) site

• Exit site (E) site

polypeptide chain

The letter N represents the amino end of the protein; C represents the carboxyl end

Initiation in Prokaryotes step 1

The three initiation factors (IF1, IF2, IF3) first bind to the small ribosomal subunit, and this complex in turn binds to mRNA (step 1)

initiation factor 3

binds the small ribosomal subunit (30s) to prevent premature binding of the large ribosomal subunit (50s)

factor 1

Initiator factor 1 binds the small subunit (30S) and prevents aminoacyl tRNA from binding to the A-site prematurely

• IF1 ensures fMet occupies P-site first and translation starts precisely at AUG

factor 2

Initiation factor 2 +GTP facilitates the binding of the initiator tRNA (fMet) with the mRNA

• Stabilizing it to the P-site

small subunit

binds the mRNA via the Shine-Dalgarno sequence

initiation complex

• Small ribosomal

• Subunit + initiation

• Factors + mRNA at codon AUG

• Combines with large ribosomal subunit

Shine-Dalgarno sequence (AGGAGG)

• Precedes AUG start codon in bacteria

• Base-pairs with region on 16S rRNA of 30S small subunit,

• Facilitating initiation

Hydrolysis of GTP

(linked to IF2) causes the dissociation of all initiation factors and allows the large ribosomal subunit to bind, forming the complete 70s initiation complex

elongation requires

• Small and large ribosomal subunits

• mRNA molecule

• GTP

• Charged initiator tRNA

• Mg2+

• Elongation factors: Tu, Ts, and G

elongation

• Both ribosomal subunits assembled with mRNA

• Forms P site and A site

• With start codon in the P site, the initiation complex is now poised for the insertion into the A site of the second aminoacyl tRNA

• Charged tRNAs are transported into the complex by one of the elongation factors (EFs)

general process of elongation

• Initiator tRNA (fMet) is in the P-site bound to AUG

• Next charged tRNAs enter the complex at the A-site

• Peptide bond is formed between the AAs in two tRNAs, catalyzed in the interface between them

• After the peptide bond is formed, the new polypeptide is transferred to the A-site tRNA (peptidyl transfer) – elongating (growing) the polypeptide chain

• Then the ribosome moves along (translocate) the mRNA by one codon

• This shifts the empty (uncharged) tRNA from the P-site to E-site (exit), where its eventually released

• Meanwhile, the tRNA carrying the new polypeptide chain moves from A-site to Psite

• This positions the next codon of the mRNA in the A-site to accept another tRNA

The three Elongation factors (EF-Tu, EF-Ts, EF-G) facilitate tRNA entering A-site, recharge GTP, and translocate subunits to next codon (step 2)

• Elongation factor Tu (EF-Tu) (EF-I in eukaryotes) bound with GTP facilitates binding of the proper charged tRNA into the A-site

• GTP is hydrolyzed, releasing EF-Tu to be recharged by EF-Ts

• Peptide bond formation between the amino acid in the P-site and amino acid in the A-site (catalyzed by the rRNA)

• Translocation in the Ribosomal subunits is aided by EF-G (EF-II in eukaryotes) and the hydrolysis of GTP -> GDP

• Release of the empty tRNA from the E-site

Translation: Elongation

23S rRNA

• In the large subunit, it is a ribozyme that catalyzes peptide bond formation between amino acid on tRNA at A site and growing peptide chain bound to tRNA in P site

• Uncharged tRNA moves to E (exit) site

• tRNA bound to peptide chain moves to P site

• Sequence of elongation and translocation is repeated over and over

• Peptidyl transferase was initially believed to be catalytic enzyme for above reactions. It is in fact the catalytic activity of 23S rRNA. Peptidyl transferase is a domain within 23S rRNA, it is a function of 23S rRNA

termination

• Signaled by stop codons (UAG, UAA, UGA) in A site

• Codons do not specify any amino acid

GTP-dependent release factors

• Stimulates hydrolysis of polypeptide from peptidyl tRNA

• released from translation complex

termination

• A release factor (RF1 or RF2) binds to the stop codon in the A-site of the ribosome

• This causes release of the polypeptide from the P-site (through hydrolysis)

• Another release factor (RF3) with GTP binds the ribosome

• Hydrolysis of GTPà GDP causes a conformational change in the ribosome

• The ribosome separates, releasing the mRNA, tRNA, and release factors

stop codons

• No tRNA enters the A site

• Ribosomal subunits dissociate and tRNA is released

• Polypeptide proceeds to fold into 3D conformation

table to protein synthesis in bacteira cells

Polysomes (or polyribosomes)

• mRNAs with several ribosomes translating at once

• As mRNA passes through ribosome, its free to associate with another small subunit

Translation in eukaryotes

• More complicated!

• Ribosomes are larger and longer lived than bacteria

• Transcription occurs in nucleus

• 5’ end of mRNA capped with 7-methylguanosine residue at maturation, which is essential for translation

• Poly-A tail added at 3’ end of mRNA

• Translation occurs in the cytoplasm – in bacteria transcription and translation are coupled due to lack of organelles

• Most eukaryotic mRNAs are translated in a cap-dependent fashion

Translation

• 5’ end of mRNA capped with 7-methylguanosine (m7G) residue at maturation, which is essential for translation

• Initiation of translation involves the m7G cap associating with the small ribosomal subunit in what is called cap-dependent translation •

• Mediated by eukaryotic initiation factors (eIFs), a unique initiator tRNA and the small ribosomal subunit

• Analogous to the Shine-Dalgarno sequence in that it sets the reading frame for translation

Kozak Sequence

• Eukaryotic mRNAs contain purines (A or G) three bases upstream from AUG initiator codon, followed by G (Kozak Sequence)

• A/GNNAUGG

• Kozak sequence is considered to increase efficiency of translation by interacting with initiator tRNA

Translation in eukaryotes

• Requires more factors for initiation, elongation, and termination than in bacteria

• Not all ribosomes are not free-floating; instead some are associated with endoplasmic reticulum

• Those that are free-floating, are translated as polyribosomes through closed loop translation

closed loop translation

• The mRNA forms a loop that is closed where the cap and tail are brought together

• Poly-A-Binding proteins bind to the cap-binding protein to form the loop

• Facilitated by initiator binding proteins and the eIF4G (cap binding protein) that serve as scaffolding proteins

• Poly A tails may contribute to initiation • Ribosome recycling

Inborn Errors of Metabolism: Connection between genes and proteins

• Early insight that proteins are important in heredity was provided by studies of diseases caused by errors in metabolism (1902)

• Several human disease appeared to have patterns of inheritance and lead researchers to conclude they were genetic in nature

• In several instances, unaffected parents were of consanguineous relations, indicating recessive traits and heterozygosity

• Concluded conditions were caused by lack of heredity information that controlled chemical reactions in the body

Errors in metabolism

• Alkaptonuria and phenylketonuria result from mutations that lead to metabolic blocks -

• Alkaptonuria: Individual cannot metabolize an enzyme called alkapton, leading to accumulation of an intermediate substance called homogentisic acid

• Phenylketonuria: Individual is unable to convert phenylalanine to tyrosine

• Hundreds of medical conditions caused by errors in metabolism due to mutant genes

Phenylketonuria (PKU)

• Phenylalanine hydroxylase is inactive in affected individuals

• Phenylalanine is not converted to tyrosine

• Patients experience a dietary buildup of phenylalanine to toxic levels; low levels of tyrosine

• Phenylalanine enters cerebrospinal fluid with elevated levels; this results in seizures and cognitive disabilities

One-Gene: One-Enzyme Hypothesis

• George Beadle showed the first evidence that genes are directly responsible for synthesis of enzymes

• Beadle and Tatum showed nutritional mutations in bread mold Neurospora caused loss of enzymatic activity E

• xperiment led to one-gene: oneenzyme hypothesis

Auxotrophy

is the inability of an organism to synthesize a particular organic compound required for its growth

mutant

knockout/loss of function

Analysis of Biochemical Pathways

• Neurospora: Metabolic pathway investigated

• Synthesis of amino acid arginine

• Seven mutant strains were studied

• Mutations prevented conversion of citrulline to arginine

• By studying these mutants, you can deduce the steps in a biochemical pathway

• If mutant fungi can grow on a nutrient, then the mutant gene must occur prior to that nutrient in the pathway

One-gene:

one-protein hypothesis changed to one gene: onepolypeptide chain hypothesis

Two factors modified the hypothesis

• Nearly all enzymes are proteins

• not all proteins are enzymes • Proteins have subunit structure with two or more polypeptide chains

Sickle-Cell Anemia

• The first direct evidence that genes specify proteins other than enzymes came from the work on mutant hemoglobin molecules derived from humans who have the disorder sickle-cell anemia

• Sickle-cell anemia

• Recessive genetic disease

• Afflicted individuals are homozygous for HbS hemoglobin allele

• Erythrocytes become elongated under low oxygen tension

• Heterozygotes are carriers of affected gene but largely unaffected

Mutant Hemoglobin Causes Sickle-Cell Anemia

• Chemical differences exist between normal and sickle-cell hemoglobin

• Two molecules designated

• : - HbA and HbS

• Fingerprinting demonstrated that HbA and HbS hemoglobins differ by a single peptide fragment

• This difference results in the sickle shape

Using starch gel electrophoresis:

• Found Hb from individuals with sick-cell anemia migrated differently in an electric field

• Migration indicated all molecules had a negative charge

• HbA Migrated further than HbS

• suggesting HbA had a greater charge

• Unsure whether a chemical change altered charge

Fingerprinting

• 2D separation of peptide fragments into a distinctive pattern of spots

• Hb digested into peptide fragments, and separated on an absorbent paper by an electric field (migration by charge)

• Paper is then turned on a right angle, placed in a solvent, migrates peptides in a second direction by chromatographic action

• resolved differences in uncharged side chains

• From this, they saw a difference of a single peptide fragment •

• Valine was substituted for glutamic acid

Protein Function

• Proteins are critical to the life of the cell

• They catalyze chemical processes, provide structure and support, transport substances, and play a role in cellular communication and defense

• Protein structure is organized like nucleic acid – primary, secondary, tertiary, and even quaternary

Polypeptides

Precursors to proteins

• Amino acids assembled on and released from ribosomes as polypeptides

• Amino acid polymer

proteins

• Polypeptides folded up into a functional three-dimensional conformation

• Wide variety of protein structures – the diversity of proteins provides the diversity of life forms

amino acids

• Central carbon atom

• Carboxyl group (COO-)

• Amino group (NH3+)

• Hydrogen (H)

• R (radical) group bound to central carbon atom

• Building blocks of protein

• 
  

• R group (side chain) of amino acid determines the physiochemical properties of type of amino acid

• Nonpolar (hydrophobic) -

• Polar (hydrophilic)

• Positively (basic) and negatively (acidic) charged

Peptide Bond:

• Dehydration (condensation) reaction facilitates bond between carboxyl group of one amino acid and amino group of another

• Two amino acids linked together constitute a dipeptide, three a tripeptide, etc.

• Proteins are built from N terminus to C terminus

Posttranslational Modifications

• Polypeptide chains modified once they have been synthesized

• Modifications are crucial to functional capability of final protein product

protein Structure: 4 levels of Protein Structure

N-terminus amino acid removed or modified

Formyl group or formylmethionine residue is removed enzymatically from peptide

Individual amino acids residues modified

• E.g. phosphorylation of tyrosine

• Change conformation or interacting proteins

Carbohydrate side chains are sometimes attached

Producing glycoproteins

Polypeptide chains may be trimmed

• Signaling sequences for membrane bound or secreted proteins, mitochondrial targeting signaling, signals are cleaved afterwards

• Signal sequences are removed

Polypeptide chains often complexed with metal

• Non-proteinaceous elements called prosthetics groups -

• Commonly vitamins, metals, metal-containing molecules (e.g. hemoglobin)

proteins folding

• Some proteins can begin to fold into their 3D conformation based on chemical properties of amino acid sequence as soon as they are translated, called cotranslational folding

• Many are nonspontaneous and dependent on chaperones: proteins that mediate folding process

• Discovered in Drosophila, where they are called heat shock proteins

• Expressed in response to high heat to ensure proper folding under these conditions

misfolded proteins

• Eukaryotes: tagged with a small covalently attached protein called ubiquitin, attached by ubiquitin ligase

• Ubiquitin is recognized by proteosome, a large protease complex, and degraded

Protein Shape/Structure is crucial to function

Protein misfolding (wrong 3D structure) or denaturation (loss of 3D structure) can lead to a loss of biological activity

Diseases of protein folding (transmissible diseases)

• Creutzfeldt-Jakob disease (disease of secondary protein structure)

• Transmittable brain disorder in mammals

• Scrapie in sheep

• - Bovine spongiform encephalopathy (BSE; mad cow disease) -

• Presence of prions (misfolded proteins) in brain that cause aggregates that can be infectious

• Altered form of a normal cellular version synthesized in neurons of the brain

• When abnormal prions contact normal proteins, it refolds the normal protein, creating clusters that eventually destroy the brain

progressive neurodegenerative

linked to abnormal protein aggregates in brain and of secondary protein structure

Diversity of Proteins

Proteins are the most diverse macromolecules found in cells and play many different roles

immunoglobulins

Function in immune system of vertebrates

transport proteins

Movement of molecules across membranes

Hormones and their receptors

: Regulate various types of chemical activity

histones

bind to dna in euk

Transcription factors

Regulate gene expression

Hemoglobin and myoglobin

Transport oxygen, which is essential for cellular metabolism

Collagen and keratin

Structural proteins associated with skin, connective tissue, and hair of organisms

Actin and myosin

Contractile proteins found in muscle tissue

Tubulin

Basis of microtubule function in mitotic and meiotic spindle fibers

enzymes

• Most diverse and extensive group of proteins

• Specialize in catalyzing chemical reactions; depends on its active site

• Increase rate of chemical reaction reaching equilibrium

• Involved in biological catalysis: Process whereby enzymes lower energy of activation for given reaction

• Rxns are either Catabolic or Anabolic

• Catabolism: degrades larger into small simpler ones, releases chemical energy

• Anabolism: synthetic phase of metabolism (builds molecules like nucleic acid, proteins, lipids, etc.)

Functional domains of proteins:

Specific sequences are associated with unique functions in the protein

• Sequences with 50-300 amino acids constitute protein domains

• - Sequences constitute protein domains that fold into stable, unique conformations -

• Different protein domains impart different functional capabilities e.g.: -

• DNA binding

• Enzyme activity

Exon Shuffling

• Hypothesis concerning genetic origin of protein domains

• Proposed collection of exons originally present in ancestral genes that were brought together via recombination during evolution – essentially exons may have been reshuffled between genes

• Proposes this was possible through retrotransposons (jumping genes) or DNA errors in homologous recombination (unequal crossover)