12 transcription and translation

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Chapter 16

How Genes Work



Genes

Genes – units of genetic information (DNA) that

carry instructions for building polypeptides

(proteins) or functional RNA molecules along with

regulatory sequences



Gene Expression

Gene Expression – process

of converting archived

information (DNA

sequences) into molecules(such as proteins) that

actually do things in the cell

DNA(information

storage)

mRNA(information

carrier)

Proteins(melanocortin

receptor)

Forest mouse

TRANSLATION

TRANSCRIPTION

Mice with this DNA sequence

have dark coats.



How Do Genes Specify the Production

of a Protein?

DNA does NOT directly catalyze protein production

reactions

DNA sequence acts as a code Carries information about how to make proteins

Eukaryotic organisms:

DNA – nucleus

Ribosomes (sites of protein production) – cytoplasm



Messenger RNA Serves as Intermediary

Between Genes and Proteins

DNA

mRNA Do mRNA

molecules

connect DNAto proteins?

mRNA

Ribosome Protein

DNA is

found

in the

nucleus

Protein

synthesis

takes place in

the cytoplasm



Central Dogma of Molecular Biology

Francis Crick (1958) – The Central Dogma of Molecular

Biology – summary of the flow of information in cells

DNAin format ion

storage

mRNA

in fo rmat ioncarrier

Proteinact ive cel l

machinery

3

5 3

5

Transcription

Translation



Central Dogma: Modifications

Many genes code for RNA molecules (rRNA and tRNA)

that do not function as mRNA and are not translated

into proteins

Transfer RNA (tRNA) – “interpreter” molecule; transfers amino

acids to the ribosomes

Ribosomal RNA (rRNA) – component of ribosomes

Information flow is not always in one direction – in

some cases, information flows from RNA back to DNA

(example: retroviruses)



DNA mRNA Protein

How does a sequence of nucleotide bases (in DNA

and then mRNA) code for a sequence of amino acids

in a protein? Transcription (DNA mRNA)

Complementary Base Pairing Rules

Translation (mRNA Protein)Genetic Code – rules that specify the relationship

between a nucleotide sequence and an amino acid

sequence



Complementary Base PairingDNA RNA

DNA Nucleotide RNA Nucleotide

Guanine (G) Cytosine (C)

Cytosine (C) Guanine (G)Thymine (T) Adenine (A)

Adenine (A) Uracil (U)



Genetic Code: A Triplet Code

There are only 4 nucleotide bases, and they must specify 20 amino acids.

How many

bases specifya single amino

acid?

5 3

mRNAmolecule

4 Bases?… 3 Bases? 2 Bases? 1 Base?

Since there are only 4 bases,

a 1-base code could specifyonly 4 amino acids.

A 2-base code could specify amaximum of 4 4 = 16 amino acids.

4 < 20 : Not enough

16 < 20 : Not enough 64 > 20 : More than enough

A 3-base code could specify a maximum

of 4 4 4 = 64 amino acids.



Genetic Code: A Triplet Code

Triplet Code – each amino acid is coded for by a

group of three bases

Codon – group of three bases that specifies a

particular amino acid



Cracking the Genetic Code

Marshall Nirenberg and Heinrich Matthaei – developmethod for synthesis of RNAs of known sequence (particularcodons)

Use known RNA sequences to determine genetic code…

Example:

AAAAAAAAAAAA lys-lys-lys-lys

UUUUUUUUUUUU phe-phe-phe-phe



The Genetic Code

Calvin distinguished alumnus FritzRottman helped decipher the genetic

code while working as a postdoc in

Marshall Nirenberg’s lab at the

University of Michigan. Nirenberg won

a Nobel Prize for this work in 1968.

http://www.nobelprize.org/nobel_prizes/medicine/laureates/1968/

http://www.nobelprize.org/nobel_prizes/medicine/laureates/1968/

http://www.calvin.edu/publications/spark/2008/fall/rottman.htm



Genetic Code

The genetic code…

is redundant – more than one

triplet may specify the same

amino acid

is unambiguous – each codon

has only one meaning

is universal – same geneticcode is used by all living things

is conservative – first two

bases of codons that specify

same amino acid are identical



Genetic Code

The genetic code…

has start and stop codons

Start Codon (AUG) –

identifies the site at

which protein synthesis

should start; codes for

methionine

Stop Codons (UAA,UAG, UGA) – signify that

protein synthesis is

complete



Using the Genetic Code

1. Transcribe and translate as much of this gene as you can,

assuming this sequence is part of a template strand ……

3’ – C G T A C C A G T T C G G A T C G C A G T – 5’

a. …near the beginning of a gene (i.e., it contains a start codon).

b. …near the end of a gene (i.e., it contains a stop codon).



Mutations

Mutation – any permanent change in an organism’s DNA

Effects? – Depend upon location of mutation

TRANSLATION

TRANSCRIPTION

DNA(information

storage)

mRNA(information

carrier)

Proteins(active cellmachinery)

3

5 3

5

TRANSCRIPTION

TRANSLATION

3

5

5

3



Mutations

Point Mutation – replacement of one nucleotide

with another

Silent Mutation – does not alter amino acid sequence

Missense (Replacement) Mutation – changes one aminoacid to another

Nonsense Mutation – changes codon for an amino acid

to STOP codon – polypeptide chain is too short – non-

functional protein

Base Insertion or Deletion (Frameshift Mutation)

– alter reading frame of mRNA triplets



Missense Mutation: Example

5

3

Mutant

5

3

Normal

DNA sequenceof non-template(coding) strand

DNA sequenceof non-template(coding) strand

Amino acidsequence

Amino acidsequence

Sickled red blood cells

Normal red blood cells



Frameshift Mutations

Insertion or deletion of bases, alters reading frame

(grouping of codons)

Affects all codons (and therefore amino acids) positioned

after site of insertion or deletion

Generally, results in non-functional protein



Mutations: Summary



Chapter 17

Transcription

& Translation



From DNA to Proteins: Gene

Expression

Gene Expression – process by which genetic information

flows from genes to proteins; the transcription and

translation of a gene; protein production

Transcription – process by

which messenger RNA

(mRNA) is made from a

DNA template

Translation – process by

which proteins and

peptides are synthesized

from mRNA



Transcription



Transcription

Transcription – process by which RNA is made

from a DNA template

catalyzed by RNA polymerase

proceeds in 5’ 3’ direction

template-directed (complementary to DNA); only one of DNA

strands is transcribed (template strand) for a particular geneNon-template(coding) strand DNA

RNA

Temp late Strand

5

5

5

3 3

3

3

5



Transcription

Non-template(coding) strand DNA

RNA

RNA

Temp late strand

5

5

5

5

3 3

3

3 DNAtemplate

3

5

3

5 Phosphodiester bond is

formed by RNA polym erase

after base pairing occurs

Hydrogen bonds form betweencom plementary base pairs

Note: RNA utilizes uracil instead of thymine;

therefore A (DNA) pairs with U (RNA)



Transcription

Where should RNA

polymerase begin

transcribing?

…at a promoter



Promoters

Promoter – short sequence of DNA that facilitates binding of

RNA polymerase; enables transcription of downstream

genes; acts as “start transcription here” signal

Promoter (on non-template strand)

+1 site

Upstream DNA Downstream DNA

Template strand of

gene to be

transcribed



Transcription: Initiation

Promoter (on non-template strand)

UpstreamDNA

Activesite

DownstreamDNA

RNA polymerase

Initiation: RNA polymerase binds to promoter



Transcription: Elongation

RNA

RNA exitsite

UpstreamDNA

Downs tream DNA(contains genetic info that is

being transcribed)

Elongation: RNA polymerase moves along DNA, synthesizing

RNA in the 5’3’ direction

RNApolymerase

5’ end



Transcription: Termination

Hairpinloop

RNApolymerase

Upstream DNA

DownstreamDNA Stop

sequence

RNA

RNA polymerase reaches a DNA stop sequence,which codes for RNA that forms a hairpin. The RNA hairpin causes the RNA strand to separatefrom the RNA polymerase, terminating transcription.

RNA

DNA

Termination: transcription stops when RNA polymerase

reaches a DNA stop sequence – codes for RNA that forms

a hairpin



Transcription



Products of Transcription

Transcribed genes may code for…

Messenger RNA (mRNA) – carries genetic

information from DNA in nucleus to

ribosomes in the cytoplasm

Transfer RNA (tRNA) – serves as

“interpreter” molecule; transfers amino

acids to the ribosomes

Ribosomal RNA (rRNA) – component of

ribosomes (along with ribosomal proteins)

Ribozymes – RNA molecules that catalyze

chemical reactions

E k ti T i ti



Eukaryotic Transcription:mRNA Processing

In eukaryotes, the mRNA transcript that

is released from RNA polymerase (pre-mRNA) is not ready to be translated; it

must first be processed…

mRNA processing occurs in thenucleus before mRNA is exported tothe cytoplasm for translation

1. Addition of 5’-cap

2. Addition of 3’-polyA tail

3. Removal of introns (self-splicing)

E k ti T i ti



5’-cap – composed of modified guanine nucleotide;

serves as a recognition signal for translation machinery

(ribosome)

3’-poly(A) tail – composed of 100-250 adenine

nucleotides; facilitates transport out of nucleus; protects

mRNA message from degradation in the cytoplasm


3 5

Poly(A) tail 5 cap

Coding region

E k ti T i ti




Eukaryotic genes (unlike prokaryotic genes) contain

noncoding regions

Exons – Expressed (coding) regions

Introns – Intervening/Interrupting (noncoding) regions

Pre-mRNA transcript:

DNA:

Promoter

Intron 1 Intron 2

Exon 1 Exon 2 Exon 3

Spliced transcript:

3

5

5

3

5

3



Removal of Introns: RNA Splicing

Splicing is catalyzed by

small nuclear RNAs –

snRNAs

Form a multiprotein

complex called a

spliceosome



Translation



Translation

Translation – process by which proteins and peptides

are synthesized from mRNA

Occurs at ribosomes

Uses transfer RNA (tRNA) as an “interpreter” molecule



Prokaryotic Translation

In prokaryotes… transcription and translation are tightly

coupled – translation begins before transcription is

complete

Ribosome translatesmRNA as it is beingsynthesized by RNApolymerase

5 endof mRNA

1

2

1 1

2

3

Protein

Ribosome

RNA polymerase

(3 end of template strand) Start of gene End of gene

(5 end of template strand)

This does not occur in

eukaryotes – why not?



Eukaryotic Translation

In eukaryotes…

transcription and

translation areseparated in space

and time

mRNA

DNA

MaturemRNA

Transcription andRNA processing

in nucleus

Mature

mRNA

Translationin cytoplasm

Protein

Ribosome



Translation

Translation between the language of nucleic acids

(nucleotides) and the language of proteins (amino

acids) requires an “interpreter” (adapter)

Aminoacids

Interpreter

molecules

mRNA

Codon Codon Codon Codon



Transfer RNA (tRNA): the “Interpreter”

tRNA structure

Cloverleaf shape due to complementary base pairing

between portions of molecule

T f RNA (tRNA) th



Transfer RNA (tRNA): the

“Interpreter”

tRNA structure

Contains amino acid binding

site (3’ end)

Contains an anticodon at

opposite end – complementary

to mRNA codon

Bind ing si te for

amino acid

Ant icodon

(b inding site for

mRNA codon)

Transfer RNA (tRNA)



There are 64 codons; 61 code for amino acids

But… there are only ~40 different tRNA molecules

Wobble Hypothesis – non-standard base

pairing (between codon and anticodon) is

acceptable in the third position as long as

it does not change the amino acid for

which the codon codes

Transfer RNA (tRNA)Wobble Hypothesis



Ribosomes: Site of Protein Synthesis

Ribosomes – site of protein synthesis; composed of

ribosomal RNA (rRNA) and proteins

Small subunit – holds mRNA in place

Large subunit – has three binding sites for tRNAs; containsactive site for peptide bond formation

protein synthesis is catalyzed by

rRNA - ribozymes

5 3 Codon

Large

subun i t

Smallsubun i t

mRNA Anticodon



Ribosomes



Ribosomes

E (exit) site - holds tRNA

that will exit (amino acid

no longer attached)

P (peptide) site - holds

the tRNA with growing

polypeptide attached

A (amino acid) site -

holds incoming tRNA

(with attached amino

acid)



Translation: Initiation



Translation: Elongation

Arrival of tRNA/amino acid - appropriate tRNA (carrying anamino acid) binds to the mRNA codon in the A site viacomplementary base pairing




Peptide bond formation – peptide chain is covalently linkedto amino acid in the A site; reaction catalyzed by a ribozyme




Translocation – ribosome moves down the mRNA (5’3’ direction)

moves empty tRNA into E site (exit site)

moves tRNA containing polypeptide into P site

open A site exposes new mRNA codon




Elongation requires energy

and assistance from

elongation factors

Multiple ribosomes can

translate a single mRNA at

one time - polyribosomes

5 3 mRNA

Ribosomes

Growing polypeptides



Translation: Termination

Stop codon is exposed in A site; release factor fills A site

Bond linking P site tRNA with polypeptide is hydrolyzed;

polypeptide is released from ribosome

Small and large subunit of ribosome and mRNA dissociate



Post-Translational Modification

Post-translational modifications – processing steps that

are required to make a protein functional; such as…

Protein folding – may require molecular chaperones

Addition of chemical groups (eukaryotes – in ER and Golgi)

Phosphorylation / dephosphorylation



Summary of Gene Expression

12 transcription and translation

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