From Gene to Protein

Campbell and ReeceChapter 17

process by which DNA directs the synthesis of proteins or RNA

synthesis of proteins 1. transcription2. translation

Gene Expression

Evidence from study of metabolic disorders:

1902: British physician 1st to suggest genes responsible for phenotype thru enzymes that catalyze specific chem rx in the cell

How Gene to Protein Figured Out

Garrod hypothesized that symptoms of an inherited disease are due to a gene that leads to inability to make a certain enzyme

1 of 1st to realize Mendel’s principle’s of heredity applied to more than pea plants

Inborn Errors of Metabolism

Signs & Symptoms: urine turns black when alkapton

(chemical in urine) reacts with air missing enzyme in pathway that

degrades phenylalanine (a.a.)

Alkaptonuria

worked with a bread mold Neurospora crassa

bombarded it with radiation (already known to cause genetic changes)

then checked for survivors who had different nutritional needs from wild-type mold

Beadle & Tatum Experiment

individually put yeast in different mediums (agar with different nutrients)

identified mutants that could not survive on minimal nutrients placed them in complete growth medium (minimal med. + all 20 a.a. + few vitamins & minerals)

Beadle & Tatum Experiment

Beadle & Tatum’s results supported their hypothesis

1958: Nobel prize

1-Gene-1-Polypeptide

revised over time: not all proteins are enzymes some proteins have >1 polypeptide now: 1- gene-1-protein hypothesis not 100%: some eukaryotic genes can

each code for a set of closely related polypeptides via alternative splicing

1-Gene-1-Polypeptide

the synthesis of RNA using information in DNA

mRNA made using complimentary base pairing

Transcription: short version

synthesis of a polypeptide using the information in mRNA

“translates” message in mRNA a.a.

Translation: short version

4 nucleotide bases to code for 20 a.a.

triplet code: 3 consecutive bases code for 1 of the a.a./ stop

The Genetic Code

during transcription: DNA helix unwound 1 strand only transcribed (could be

either side depending on the gene)

Template Strand

uracil added as compliment to adenine

ribose as its 5-carbon sugar single stranded

mRNA

nucleotide triplets of DNA or mRNA that specifies a particular amino acid or termination signal

basic unit of the genetic code written in 5’ 3’ direction (in DNA 3

bases read in 3’ 5’ direction)

Codons

Genetic Code

early 1960’s Nirenberg: synthesized mRNA using

only uracil (UUUUUUU…) added it to test tube with all 20 a.a.,

ribosomes translated into polypeptide made up of

phenyalanine now knew UUU = Phe did same for AAA= Lys, CCC = Pro, GGG

= Gly

Cracking the Code

all 64 a.a. deciphered by mid-1960’s 3 codons code for “stop” marking

end of translation AUG functions as “start” & Met

Met may or may not be clipped off later

Cracking the Code

>1 triplet codes for each of the a.a. but any 1 triplet codes for only 1 a.a

redundant triplets usually only differ in the 3rd base

Genetic Code is Redundant

translating the code in correct groupings

example: Did the red dog eat the bug?

Idt her edd oge att heb ug?

Reading Frame

Reading Frame

code is nearly universal: bacteria complex multicellular

organisms CAU = His insert genes into other species & get

same result (human insulin gene in bacteria)

exceptions: certain unicellular eukaryotes & in organelle genes of some species

Evolution of Genetic Code

unwinds 2 strands of DNA binds nucleotides together as build

mRNA only in 5’ 3’ direction (like DNA

polymerase)

RNA Polymerase

1. Initiation2. Elongation3. Termination

3 Stages of Transcription

After RNA polymerase binds to promoter, ¤ DNA strands unwind polymerase begins RNA synthesis @

start pt. on template strand

Initiation

promoter: usually includes w/in it the transcription start point (a nucleotide where transcription begins) & extends several dozen or more nucleotide pairs upstream from start pt.

RNAP can assemble nucleotides only in 5’ 3’ direction (just like DNA polymerase)

unlike DNAP, RNAP does not require a primer

Initiation

nucleotide where RNA synthesis actually begins

RNAP binds in precise location & orientation on the promoter where determines where transcription starts & which of the 2 strands will be transcribed

Start Point

Bacteria: 1 single RNAP used to make all types

RNA Eukaryotic Cells:

@ least 3 types RNA polymerase II used for RNA synthesis I and III used to transcribe RNA not

used for protein synthesis

RNA Polymerase

Prokaryotes : RNAP recognizes & binds to the

promoter by itself Eukayotes:

collection of proteins , transcription factors, mediate the binding of RNAP & initiation of transcription

RNA Polymerase

must 1st attach to promoter b/4 RNAP II can bind to it RNAP II + transcription factors =

Transcription Initiation Complex TATA box: DNA sequence in

eukaryotic promoters crucial in forming the transcription initiation complex

Transcription Factors

RNAP moves downstrean, unwinding the DNA & elongating the RNA transcript 5’ 3’

~ 10 – 20 nucleotides exposed in wake of transcription the 2 DNA

strands spontaneously rewind length of DNA transcribed =

transcription unit

Elongation

Elongation

mechanism differs between prokaryotes & eukaryotes

Bacteria: transcription proceeds thru terminator sequence in the DNA the transcribed RNA functions as the terminator sequence causing RNAP to detach

prokaryotes have no further modification

Termination

RNAP II transcribes a portion of DNA called the polyadenylation signal (AAUAAA) in the pre-mRNA

~10 – 35 nucleotides downstream from that sequence proteins ass’c with transcription cut the pre-mRNA free from the polymerase

pre-mRNA then modified

Termination in Eukaryotes

in eukaryotes only both ends of primary transcript

altered certain interior sections cut out &

remaining parts spliced back together

RNA Processing

5’ end receives a 5’cap: modified G is added after ~ 20 – 40 nucleotides in mRNA

3’ end modified: enzyme adds 50 -250 A’s to the AAUAAA forming a poly-A tail

mRNA Ends

1. facilitate exit of mRNA from nucleus

2. protect mRNA from degradation of hydrolytic enzymes

3. help ribosomes attach to the 5’ end

Functions of Modified Ends of mRNA

cut-and-paste job removing segments of RNA that were transcribed

average size transcript: 27,000 nucleotides

average size protein: 1,200 nucleotides (400 a.a.)

RNA Splicing

noncoding, intervening sequence w/in primary transcript that is removed from the transcript during RNA processing; also refers to the region of DNA from which this sequence was transcribed

Introns

sequence w/in primary transcript that remains in the RNA after RNA processing; also refers to the region of DNA from which this sequence was transcribed

Exons

signal: short nucleotide sequence @ each end of an intron

particle called “snurp” recognizes splice sites small nuclear ribonucleoproteins

(snRNP’s) in nucleus made of RNA + protein small nuclear RNA ~150 nucleotides

RNA Splicing

combination of several different snRNP’s (almost size of ribosome)

interact with certain sites along intron releasing intron rapidly degraded

then joins ends of exons together

Spliceosome

RNA Splicing

RNA molecules that function like enzymes in some organisms

intron RNA can act like ribozyme & catalyze its own excision

Ribozymes

3 properties of RNA enables some RNA molecules to function as enzymes:

1. single-stranded: 1 sequence can interact w/another using base pairing

2. some of bases contain functional groups (like a.a) that could participate in catalysis

3. ability to form H-bonds adds specificity

Ribozymes

RNA

still having debate about importance of introns & RNA splicing in evolution

they both have adaptive benefits do not know functions of most

introns

Importance of Introns

single gene can encode >1 kind of polypeptide

know many genes that make 2 or more different polypeptides depending on what was removed as introns during gene splicing

called: alternative RNA splicing

Importance of Introns

Drosophila sex differences due to how RNA transcript is spliced

Human Genome Project: 1 of reasons humans get by with same # genes as a nematode

Alternative RNA Splicing

tRNA: transfers a.a. from cytoplasmic pool of a.a to ribosome where it’s a.a. is added to polypeptide chain

cell keeps supply of all 20 a.a. on hand degradation of other molecules synthesizes them using building blocks

in cytoplasm

Translation: Closer Look

brings specific a.a to ribosome 1 end has a.a./ other end has anticodon

which H-bonds with codon on ribosome tRNA translates the codes into the

corresponding a.a. tRNA is transcribed from DNA

templates & used repeatedly tRNA made of ~80 nucleotides long

with some regions folded back on self due to base pairing

tRNA

tRNA Structure

3’ end: a.a. attached

opposite end: anticodon

tRNA Structure

requires 2 instances of molecular recognition:

1. tRNA that binds to particular a.a. correct match made by group enzymes

called aminoacyl-tRNA synthetases: their active site fits only 1 of the 20 a.a.

2. pairing of tRNA anticodon with mRNA codon

Accurate Translation

~ 45 different ones (not 61 like genetic code would suggest) possible because pairing the 3rd base of

codon & 3rd base of anticodon: relaxed base pair rules

U can pair with A or G in 3’ end of codon (3rd position)

called a “wobble”

tRNA Wobble

subunits made in nucleolus rRNA transcribed & added to proteins

imported from cytoplasm ribosomal subunits cytoplasm, join

only when translating mRNA subunits ~1/3 protein & 2/3 rRNA

bacteria: 3 molecules rRNA eukaryotes: 4 molecules rRNA

Ribosomes

eukaryotic ribosomes slightly larger than prokaryotic ones

pharmaceutical products (antibiotics) designed to inactivate bacterial ribosomes that have no effect on ours Tetracyclines Streptomycin

Ribosome Structure

4 binding sites: (1st for mRNA, others for tRNA)

1. mRNA binding site2. P site: peptidyl-tRNA holds the

tRNA carrying the growing polypeptide chain

3. A site: aminoacyl-tRNA holds tRNA carrying next a.a to be added

4. E site: exit, where discharged tRNAs leave ribosome

Ribosome Structure

holds tRNA & mRNA in close proximity & catalyzes the formation of new peptide bond holding the 2 a.a together adding to carboxyl end of last a.a. in growing polypeptide chain

peptide chain passes thru exit tunnel in large subunit as it grows longer

Ribosome

3 Stages:1. Initiation2. Elongation3. Termination

Translation

small ribosomal subunit attaches to mRNA

downstream from this attachment is the start codon AUG

tRNA with UAC (Met) binds to it large ribosomal subunit attaches

(1GTP) initiation factors (proteins) required

to bring it all together

Initiation

1. Codon recognition anticodon of incoming tRNA w/c’ base 1 GTP increases accuracy & efficiency

2. Peptide bond formation part of rRNA catalyzes reaction amino end of newest a.a + carboxyl end of

peptide chain transferring pep. chain to tRNA @ A site

3. Translocation ribosome moves so tRNA @ A site P site 1 GTP

Elongation

1. ribosome reaches stop codon the A site accepts a “release factor” (shaped like tRNA but does not have aminoacyl part)

2. promotes release of bond between P site, mRNA, & last tRNA

3. 2 ribosomal subunits & ass’c proteins come apart

Termination

http://bcs.whfreeman.com/thelifewire/content/chp12/1202003.html

http://highered.mcgraw-hill.com/olcweb/cgi/pluginpop.cgi?it=swf::535::535::/sites/dl/free/0072437316/120077/micro06.swf::Protein%20Synthesis

Animation Time!

http://www.wiley.com/college/boyer/0470003790/animations/translation/translation.htm

Try at home: interactive

1 ribosome can make polypeptide of average size: 1 min

typically many ribosomes are translating a single mRNA @ given time

1st ribosome gets far enough past start codon 2nd ribosome can get started

allow cell to make many copies of polypeptide very quickly

Polyribosomes

Polyribosomes

as polypeptide chain grows longer from ribosome it will spontaneously start to fold & coil as result of a.a side chain interactions

genes determine 1’ structure which then determines 2’, 3’ and 4’ structures

Primary Structure

proteins that help with the folding

Chaperonins

additional steps that may be required b/4 protein can do its job attachment of sugars, lipids, phosphate

groups to a.a enzymatic removal of 1 or more a.a.

from leading end (amino end)

Post-Translational Modifications

Modification of Insulin

free ribosomes make proteins used in cytoplasm

bound ribosomes (RER) attached to cytosolic side while polypeptide being released into endomembrane system

both have identical small & large subunits

Targeting Polypeptides Specific Locations in Cell

Ribosomes

growing polypeptide cues ribosome to attach to ER

polypeptides of proteins destined for endomembrane system have signal peptide: sequence of ~20 a.a. at or near leading end (N-terminus) is recognized by a protein-RNA complex called signal-recognition particle or SRP

Signal Peptide

escorts ribosome to receptor protein on ER membrane

receptor part of multiprotein translocation complex

ribosome continues to make polypeptide which enters ER thru protein pore

signal protein usually removed by enzyme

SRP

use other signal peptides for protein destined for chloroplast, mitochondria, or interior of nucleus

in these, proteins made in cytosol then to organelle

signal proteins target or “address” proteins for secretion or to cellular locations used by prokaryotes too

Proteins Organelles

ultimate source of new genes large scale mutations

chromosomal rearrangements: chap. 15 small scale mutations

1 or a few nucleotide bases changed

Mutations

http://www.bodrum-hotels.com/gene-mutations/gene-mutations-and-proteins-worksheet.html

Try @ Home

changes in single nucleotide pair if occurs in gamete or cell that gamete

will be passed on to offspring if mutation has adverse effect on

phenotype is called a genetic disorder or hereditary disease

if mutation causes organism to die before fully developed it is said to be lethal

if mutation results in no change in phenotype is said to be silent

Point Mutations

Sickle Cell Anemia

point mutation dominant possible cause of

sudden death of young athletes

Familial Cardiomyopathy

replacement of 1 nucleotide pair by another pair: a few will improve activity of protein it is coding for but most will be detrimental

some silent due to redundancy of genetic code

if changes 1 a.a. for another called missense mutation if substituted a.a. similar to real one no effect some substitutions will have major

consequences

Substitutions

Nucleotide-Pair Substitutions

1. Silent2. Missense:

most substitutions in this category

3. Nonsense: substitution changes from 1 a.a. stop codon

resulting polypeptide is shorter nearly all nonfunctional proteins

Nucleotide-Pair Substitution

(+) or (-) of nucleotide pairs in a gene

disastrous effects may alter reading frame triplet

codon shifts on mRNA called frameshift mutation

whenever insertion or deletion not in a multiple of 3

if not causes major missense

Insertions & Deletions

any chemical or physical agent that interacts with DNA & can cause a mutation

1920’s: Muller used x-rays to make mutant Drosophila & he discovered it does same in humans

mutagenic radiation includes: UV radiation cause thymine dimers in DNA

Mutagens

Thymine Dimers

nucleotide analogs similar to normal DNA nucleotides insert self into DNA

Chemical Mutagens

some cause chemical changes in bases that changes their pairing properties

Chemical Mutagens

How Mutagens Determined

Gene Expression in 3 Domains

some in gene expression among eubacteria, archaea, and eukaryotes

if no nucleus: translation can begin b/4 transcription is over

Archaea show similarities to Eubacteria and eukaryotes in processes of gene expression

Differences

region of DNA whose final functional product is either a polypeptide or an ENA molecule

What is a Gene?