abbott - top 13: protein synthesis

Sir archibald garrod

studied metabolic disease - alkaptonuria

his work provided first evidence of a specific pathway btwn genes, enzymes, and metabolism

cause: two mutated copies of a gene that normally encodes for the enzyme that breaks down homogenitistic acid

(mutated versions are either not produced or done so at low levels)

research: provides that genes code for the sequence of amino acids in proteins

alkaptonuria = inherited trait

Beadle + Tatum - Experiments

experiments of Beadle + Tatym > bread mold neurospora showed a direct relation between genes and enzymes

grew nutritional mutants (auxotrophs) on a minimal medium supplemented with a single amino acid (ex. arginine)

beadle + tatum hypotheis

hypothesis: each auxotrophic strain had defect in gene that codes for an enzyme needs to synthesize a particular amino acid (ONE GENE-ONE ENZYME HYPOTHESIS)

later updated to: ONE GENE-ONE POLYPEPTIDE HYPOTHESIS

how we went from gene > polypeptide

Francis Crick gave name CENTRAL DOGMA

- flow of info from DNA > RNA > Protein

transcription + translation

transcription

mechanism where info encoded in DNA template strand is copied into complementary RNA strand

RNA polymerase copies DNA sequence of gene into RNA sequence - PROTEIN-CODING GENE is transcribed into MESSENGER RNA

translation

info encoded in RNA copy is used to assemble amino acids into a polypeptide

mRNA associates with ribosome - where amino acids specific by mRNA are joined one by one to form a polypeptide encoded by gene

some genes don't encode a polypeptide > encode variious molecules that function in transcription, translation, and other processes in the cell

transcription in prokaryotes vs. eukaryotes

eukaryotes:

- in nucleus

- produces a precursor-mRNA that must be altered to generate the functional mRNA

-preMRNA ends = modified + extra segments = removed by RNA processing

- functional mRNA exists nucleus and is translated in cytoplasm

(DNA > transcription > pre-mRNA > rna processing > mRNA > translation)

prokaryotes

- in cytoplasm

- produces functional mRNA directly (no modifications)

(DNA > transcription > mRNA > translation)

DNA + RNA nucleotides

DNA "Alphabet" - adenine (A), thymine (T), guanine (G), and cytosine (C)

RNA alphabet - A, U, G, C

sequence of RNA nucleotides in mRNA is translated into a polypeptide containing 20 diff amino acids

genetic code

genetic code: nucleotide info that specifies the amino acid sequence of a polypeptide

to code for 20 diff amino acids > four bases used in mRNA are used in combos of 3

each 3-letter word of code = CODON

- 64 letter combos

- 61 sense codons > specify amino acids

codons in DNA are transcribed into complementary RNA codons

start codon/initiator codon: AUG (specifies methionine) - always first codon read in mRNA translation

stop codon: UAA, UAG, UGA

- don't code for amino acids + stop polypeptide synthesis

amino acids specified by single codon

only two amino acids (methionine + tryptophan) - have ONLY one codon

3 key features of genetic code

1. degenerate (amino acids are coded for by more than one codon (except for methionine + tryptophan)

2. genetic code is commaless

3. genetic code is universal (same in all living organisms + viruses)

transcription: DNA directed RNA synthesis

gene consists of two main parts:

1. promoter (control sequence for transcription)

2. transcription unit (section of gene that is copied into an RNA molecule)

three stages of transcription:

1. initiation

2. elongation

3. termination

transcription: INITIATION

molecular machinery assembles at the promoter and begins synthesizing an RNA copy of a gene

machinery includes:

- transcription factors (TFs) > bind to promoter in the area of a special sequence known as TATA Box

- RNA polymerase - enzyme that catalyzes the assembly of RNA nucleotides into an RNA strand

Steps:

1. DNA is unwound to expose template strand

2. RNA polymerase II begins RNA synthesis

3. RNA made in 5>3 directino using 3>5 DNA template strand

*** ADENINE IN TEMPLATE MEANS URACIL IN RNA STRAND

transcription: elongation

RNA polymerase II moves along the gene > extends the RNA chain

DNA continues to unwind ahead of enzyme

transcription: termination

RNA transcript and RNA polymerase II are released from DNA template

differences btwn DNA replication + transcription

only one of two DNA nucleotide strands acts as a template for synthesis of complementary copy

only sequence encoding a single gene is copied

RNA polymerases catalyze the assembly of RNA nucleotides into an RNA strand

RNA molecules are single polynucleotide chains

Uracil (U) pairs with adenine (A)

differences in transcription in eukaryotes and bacteria

sequences in promoter where transcription apparatus assembles differ

eukaryotes:

- RNA polymerase can't bind directly to DNA

- need TFs to bind to promoter

- no sequences to end transcription of gene

prokaryotes:

- RNA polymearse binds directly to DNA

- specific dna sequences (TERMINATORS) end transcription of gene

transcription of non-protein-coding genes

noncoding RNA genes = genes encoding RNAs that are not translated

in eukaryotes:

- RNA polymerase III transcribes tRNA genes + gene for one of four rRNAs

- RNA poly I transcribes genes for three other rRNAs

promoters for noncoding RNA genes are specialized for the correct RNA polymerase type

prokaryotes:

- single RNA polymerase transcribes all genes

mRNA production in eukaryotes

mRNAs contain regions that code for proteins + noncoding regions that are important in protein synthesis

coding region:

- flanked by untranslated ends:

- 5' untranslated region (5' UTR)

- 3' untranslated region (3' UTR)

eukaryotic protein-coding gene is transcribed into a precursor-mRNA (premRNA) that must be processed in the nucleus to produce the translatable mRNA

modifying pre-mRNA ends

5' cap at end of 5' of preMRNA

(consists of guanine-containing nucleotide that is reversed > 3'OH group faces beginning of molecule)

capping enzyme adds 5; cap to pre-mRNA after RNA polymerase II begins transcription

5' cap (connected by three phosphate groups) = site where ribosomes attach mRNAs at start of translation

proteins bind to the POLYADENYLATION SIGNAL transcribed near the 3' end of the pre-mRNA and cleave the pre-mRNA downstream of the sequence

poly(A) polymerase adds a chain of 50-250 adenine nucleotides (=poly(a) tail) to the 3' end of the pre-mRNA

poly(A) trail protects premRNA from attack by RNA-digesting enzymes in cytoplasm

introns + exons

pre-mRNA for eukaryotic protein-coding gene contains one or more protein coding sequences = INTRONS

- removed during processing in nucleus

amino acid-coding sequences that are retained in finished mRNAs are called EXONS

- these read continuously in mRNAs without interruptions

mRNA splicing

occurs in nucleus

removes introns from premRNAs and joins exons together

takes place in SPLICEOSOME (formed btwn premRNA and several small RIBONUCLEOPROTEIN PARTICLES (snRNPs) > each consist of a short small nuclear RNA (snRNA) bound to a # of proteins

spliceosome cleaves the premRNA precisely to relase the intron > joins the flanking exons

alternative splicing

many pre-mRNAs are processed by reactions that jion exons in diff combos > produce diff mRNAs from single gene

increases # + variety of proteins encoded in cell nucleus w/out increasing size of genome

ex. a-tropomyosin gene = alternatively spliced in smooth muscel, skeletal muscle, fibroblast, liver and brain

exon shuffling

intro-exons junctions > often fall at points dividing major functional regions in encoded proteins

functional divisions may have allows for exon shuffling evolution > process where protein regions + domains are mixed into many combos

evolution > produces changes more quickly + efficiently than by alternations in individual amino acids at random pts

genome-wide analysis and human transcriptome

ENCODE = research project started by US national human genome research institute

reported most complete info about human transcriptome to date

study revealed: pervasive transcription of a genome and variable expression of GENE ISOFORMS (protein-coding genes that are transcribed to diff forms of mRNAs)

translation - tRNAs

transfer RNAs bring amino acids to ribosome > joined into polypeptide chain > determined by codons

mRNA read from 5 to 3 end > peptide assembled from N terminal to C terminal end

tRNAs > wind into 4 double-helical segments > forms cloverleaf pattern

at one end = anticodon (3 nucleotide segemtn that base pairs with codon in mRNAs) > at other end it links to amino acid corresponding to anticodon)

ex. Anticodon 3′-UCA-5′ base pairs with 5′-AGU-3′, serine (Ser)

wobble hypotheis

francis crick

pairing of anticodon with 1st two nucleotides = PRECISE

Third nucleotide = more flexibility in pairing

same tRNA anticodon can read codons that have either U or C in third position

Inosine (purine) > allows even more extensive wobble by allowing the tRNA to pair with codons that have either U, C, or A in the third position

aminoacylation

addition of correct amino acid by tRNA (aminoacylation or charging) > makes AMINOACYL-tRNA

20 diff enzymes (aminoacyl-tRNA synthetases) > 1 for each amino acid > catalyze aminoacylation

process adds free energy as the aminoacyl - tRNAs are formed

ribosomes

ribonucleoprotein particles that translate mRNA into chains of amino acids

in eukaryotes - ribosomes are either suspended freely in cytoplasm or attached to the endoplasmic reticulum

finished ribosome is made up of one large ribosomal subunit + one small ribosomal subsuit > each composed of RIBOSOMAL RNA (rRNA) and ribosomal proteins

mRNA passes through a groove in the ribosome

tRNAs interact with mRNA at 3 binding sites on ribosome:

- aminoacyl-tRNA carrying the next amino to be added to polypep binds to A SITE (AMINOACYL SITE)

-tRNA carrying growing chain binds to P SITE (PEPTIDYL SITE)

-tRNA without an amino binds to the E SITE (exit site) > before exiting ribosome

three stages of translation

initiation: components assemble on the start codon of mRNA

elongation: assembled complex reads codons (one @ time) > while joining specified aminos to pep

termination: complex disassembled after last amino specified by mRNA has been added to polypep

translation initiation

if eukaryotes > each step is aided by initiation factors (IFs)

in bacteria > small ribosomal subunit, initiator Met-tRNA, GTP, and IFs bind DIRECTLY to mRNA > directed by RIBOSOME BINDING SITE

initiator = tRNA-AUG pairing establishes the correct reading frame

translation elongatino

aminoacyl-tRNA binds to codon at A site of ribosome > facilitated by a protein ELONGATION FACTOR (EF)

peptide bond forms btwn C-terminal of growing polypep on the P site tRNA and the amino acid on the A site tRNA > catalyzed by PEPTIDYL TRANSFERASE

ribosome translocates to next codon

empty tRNA is released from E site > ribosome ready to begin the next round of elongation cycle

translation termination

takes place when A site arrives at one of stop codons on the mRNA (UAG, UAA, UGA)

stop codon is read by protein release factor (RF or TERMINATION FACTOR)

similar in prokary + eukarys

polysomes

once first ribo has begun translation > another can assemble as soon as there is room on the mRNA

ribos continue to attach as translation continuous and become spaced along the mRNA like beads on a string > forming a polysome

in prokarys, transcription + translation = coupled

polysome forms while mRNA is being made

processing polypeptides

includes removal of certain amino acids from poly chain + addition of carb + lipid group

many proteins require helper proteins (CHAPERONES/CHEPRONINS) > to fold into final functional 3D shapes

some are processed as initial inactive form (like pepsinogen) > later activated by removal of a segment of the amino acid chain

finished proteins are sorted

finished proteins sorted within cell to 3 types of compartments:

- cytosol

- endomembrane system (endoplasmic reticulum (ER), golgi complex, lysosomes, secretory vesicles, nuclear envelope + plasma membrane)

- membrane-bound organelles other than endomembrane sysem > mitochondria, chloroplasts, microbodies + within nucleus

protein sorting to cytosol

those that function in cytosol are synthesized by FREE RIBOSOMES in cytosol

these polypeps are simply released from ribosomes once translation completed

ex. microtubule proteins + glycolysis enzymes

protein sorting to endomembrane system

begin synthesis on free ribos in cytosol

amino acid SIGNAL SEQUENCE near N-terminal ends

polypep enters lumen of rough ER while attached to ribosome (COTRANSLATIONAL IMPORT)

proteins fold into final form + are "tagged" for destination (ER or golgi)

going to golgi? > further modified, packed in vesicles + delivered to lysosomes, plasma membrane + secreted from cell

protein sorting to other organelles

proteins sorted to mitochondria, chroloplasts, microbodies + nucleus are made on free ribosomes

- posttranslational import (process where protein is fully synthesized by ribosome and then imported into organelle)

- amino acid transit sequences near N-terminal ends interact with organelle specific transport compelxes in membrane of appropriate organelle

- once inside > transit peptidase removes the transit sequence

- proteins sorted to the nucleus have NUCLEAR LOCALIZATION SIGNALS (a short amino acid sequence that directs proteins to be transported from the cytoplasm into the cell nucleus)> remain with protein