Protein Synthesis

5b. Students know how to apply base-pairing rules to explain precise copying of DNA during semiconservative replication and transcription of information from DNA into mRNA.4a Students know the general pathway by which ribosomes synthesize proteins, using tRNAs to translate genetic information in mRNA.4b Students know how to apply the genetic code rules to predict the sequence of amino acids from a sequence of codons in RNA

Replication

•DNA double helix unwinds•DNA now single-stranded•New DNA strand forms using complementary base pairing (A-T, C-G)•Used to prepare DNA for cell division•Whole genome copied/replicated

The “Central Dogma”

Flow of genetic information in a cell How do we move information from DNA to

proteins?transcription

translation

replication

protein

RNA

DNA

trait

DNA gets all the glory,

but proteins do all the work!

mRNA

Where?

DNAtranscription

nucleus cytoplasm

aa

aa

aa

aa

a

aa

a

a

a

aa

proteintranslation

ribosome

trait

Transcription and Translation: An Overview (aka the Central Dogma)

Transcription

Translation

Transcription

From DNA nucleic acid languageto RNA nucleic acid language

Transcription RNA forms base

pairs with DNA C-G A-U

Primary transcript- length of RNA that results from the process of transcription

Messenger RNA mRNA- type of

RNA that encodes information for the synthesis of proteins and carries it to a ribosome from the nucleus

Transcription Making mRNA

transcribed DNA strand = template strand untranscribed DNA strand = coding strand▪ same sequence as RNA

synthesis of complementary RNA strand enzyme▪ RNA polymerase

template strand

rewinding

mRNA RNA polymerase

unwinding

coding strand

DNAC C C

C

C

C

C

C

C CC

G

G G G

G GG G

G

G

GAA

A A A

AA

AA

A A

A

ATT T

T

T

T

T

T

T T

T

T

U U

5

35

3

3

5build RNA 53

RNA polymerase RNA polymerase-

Produce primary transcript by stringing together the chain of RNA nucleotides

Which gene is read? Promoter region

binding site before beginning of gene TATA box binding site binding site for RNA polymerase

& transcription factors

Enhancer region binding site far

upstream of gene▪ turns transcription

on HIGH

Transcription Factors Initiation complex

transcription factors bind to promoter region▪ suite of proteins which bind to DNA▪ hormones?▪ turn on or off transcription

trigger the binding of RNA polymerase to DNA

Matching bases of DNA & RNA

Match RNA bases to DNA bases on one of the DNA strands

U

A G GGGGGT T A C A C T T T T TC C C CA A

U

UU

U

U

G

G

A

A

A C CRNA

polymerase

C

C

C

C

C

G

GG

G

A

A

AA

A

5' 3'

Eukaryotic genes have untranscribed regions! Eukaryotic genes are not continuous

exons = the real gene▪ expressed / coding DNA

introns = the junk▪ inbetween sequence

eukaryotic DNA

exon = coding (expressed) sequence

intron = noncoding (inbetween) sequence

intronscome out!

mRNA splicing

eukaryotic DNA

exon = coding (expressed) sequence

intron = noncoding (inbetween) sequence

primary mRNA

transcriptmature mRNA

transcript

pre-mRNA

spliced mRNA

Post-transcriptional processing eukaryotic mRNA needs work after transcription primary transcript = pre-mRNA mRNA splicing▪ edit out introns

make mature mRNA transcript

~10,000 bases

~1,000 bases

A A A A A3' poly-A tail

mRNA

5'5' cap

3'

G PPP

50-250 A’s

More post-transcriptional processing

Need to protect mRNA on its trip from nucleus to cytoplasm enzymes in cytoplasm attack mRNA▪ protect the ends of the molecule▪ add 5 GTP cap▪ add poly-A tail

▪ longer tail, mRNA lasts longer: produces more protein

Transcription is done…what now?

Now we have mature mRNA transcribed from the cell’s DNA. It is leaving the nucleus through a nuclear pore. Once in the cytoplasm, it finds a ribosome so that translation can begin.

We know how mRNA is made, but how do we “read” the code?

Translation

From nucleic acid languageto amino acid language

Translation

Second stage of protein production mRNA is on a ribosome

Reading the DNA code

Every 3 DNA bases pairs with 3 mRNA bases

Every group of 3 mRNA bases encodes a single amino acid

Codon- coding triplet of mRNA bases

AUGCGUGUAAAUGCAUGCGCC

mRNA

mRNA codes for proteins in triplets

TACGCACATTTACGTACGCGG

DNA

AUGCGUGUAAAUGCAUGCGCC

mRNA

MetArgValAsnAlaCysAla

protein

?

codon

Translation

Second stage of protein production mRNA is on a ribosome tRNA brings amino acids to the

ribosome

The code

Code for ALL life! strongest support for a

common origin for all life Code is redundant

several codons for each amino acid

3rd base “wobble”

Start codon AUG methionine

Stop codons UGA, UAA,

UAG

Why is thewobble good?

How are the codons matched to amino acids?

TACGCACATTTACGTACGCGGDNA

AUGCGUGUAAAUGCAUGCGCCmRNA

amino

acid

tRNA anti-codon

codon

5 3

3 5

3 5

UAC

MetGCA

ArgCAU

Val

Transfer RNA structure “Clover leaf” structure

anticodon on “clover leaf” end amino acid attached on 3 end

Ribosomes

Facilitate coupling of tRNA anticodon to mRNA codon

Structure ribosomal RNA (rRNA) & proteins 2 subunits▪ large▪ small E P A

Ribosomes

Met

5'3'

UUA C

A G

APE

A site (aminoacyl-tRNA site) holds tRNA carrying next amino acid to

be added to chain P site (peptidyl-tRNA site)

holds tRNA carrying growing polypeptide chain

E site (exit site) emptytRNA

leaves ribosome from exit site

Building a polypeptide

Initiation mRNA, ribosome subunits, initiator

tRNA come together Elongation

adding amino acids based on codons Termination

STOP codon = Release factor

123

Leu

Leu Leu Leu

tRNA

Met MetMet Met

PE AmRNA5' 5' 5' 5'

3' 3' 3' 3'U UA AAACC

CAU UG GGU UA AAACC

CAU UG GGU UA AAAC

CC

AU UG GGU UA AACCAU UG G

G AC

Val SerAla Trp

releasefactor

A AACCU UGG 3'

Protein targeting Signal peptide

address label

Destinations: secretion nucleus mitochondria chloroplasts cell

membrane cytoplasm etc…

start of a secretory pathway

Can you tell the story?

DNA

pre-mRNA

ribosome

tRNA

aminoacids

polypeptide

mature mRNA

5' GTP cap

poly-A taillarge ribosomal subunit

small ribosomal subunit

aminoacyltRNAsynthetase

E P A

5'

3'

RNA polymerase

exon introntRNA

AAAAAAAAGTP

20-30b

3'

promoter transcriptionstop

transcriptionstart

introns

Processing

transcriptional unit (gene)TAC ACT

DNA

DNATATA5'RNA

polymerase

pre-mRNA5' 3'

translationstart

translationstop

mature mRNA5' 3'

UTR UTR

exonsenhance

r1000+b

Protein Synthesis in Prokaryotes

Bacterial chromosome

mRNA

Cell wall

Cellmembrane

Transcription

Psssst…no nucleus!

Prokaryote vs. Eukaryote genes

Prokaryotes DNA in cytoplasm circular

chromosome naked DNA

no introns

Eukaryotes DNA in nucleus linear

chromosomes DNA wound on

histone proteins introns vs. exons

eukaryoticDNA

exon = coding (expressed) sequence

intron = noncoding (inbetween) sequence

intronscome out!

Transcription & translation are simultaneous in bacteria DNA is in

cytoplasm no mRNA

editing ribosomes

read mRNA as it is being transcribed

Translation in Prokaryotes

Translation: prokaryotes vs. eukaryotes

Differences between prokaryotes & eukaryotes time & physical separation between

processes▪ takes eukaryote ~1 hour

from DNA to protein no RNA processing

Mutations Point mutations

single base change ▪ silent mutation▪ no amino acid change▪ redundancy in code

▪ missense▪ change amino acid

▪ nonsense▪ change to stop codon

When do mutationsaffect the nextgeneration?

Point mutation lead to Sickle cell anemiaWhat kind of mutation?

Missense!

Mutations Frameshift

shift in the reading frame▪ changes everything

“downstream” insertions▪ adding base(s)

deletions▪ losing base(s)

Where would this mutation cause the most change:

beginning or end of gene?

Transcription vs. Translation Review

Transcription Process by which

genetic information encoded in DNA is copied onto messenger RNA

Occurs in the nucleus

DNA mRNA

Translation Process by which

information encoded in mRNA is used to assemble a protein at a ribosome

Occurs on a Ribosome

mRNA protein