Where are we?

We covered ……

REPLICATION now..

TRANSCRIPTION +

TRANSLATION

Figure 14.4

Nuclear envelope

Pre-mRNA

mRNA

DNA

RNA PROCESSING

TRANSCRIPTION

TRANSLATION

Polypeptide

Ribosome mRNA

DNA TRANSCRIPTION

TRANSLATION

Polypeptide

Ribosome

Which is a Bacterial cell? Which is a Eukaryotic cell? What is transcription? What is translation?

We are going to focus mostly on Eukaryotic cell transcription and translation.

Figure 14.5

DNA template strand

Protein

mRNA

3ʹ

Trp

TRANSCRIPTION

TRANSLATION

Amino acid

Codon

5ʹ

3ʹ 5ʹ

3ʹ

5ʹ

Phe Gly Ser

G U G U U U G G U C C A

C A C A A A C C A G G T

G T G T T T G G T C C A

Lets make some mRNA! In Eukaryotes we actually we first make “pre-mRNA” or “an RNA transcript” or a “primary RNA transcript” or an “immature RNA transcript”

Figure 14.9

Transcription factors

TATA box

Promoter Nontemplate strand

Start point

Transcription initiation complex forms.

Transcription initiation complex

DNA

RNA transcript

A eukaryotic promoter

Several transcription factors bind to DNA.

3ʹ 5ʹ

5ʹ 3ʹ 3ʹ 5ʹ

3ʹ 5ʹ

3ʹ 5ʹ

3

2

1 Template

Transcription factors

RNA polymerase II

3ʹ 5ʹ

3ʹ 5ʹ T A T A A A A

A T A T T T T

Transcription starts at a start point within a big sequence called the promoter.

Transcription unit=whole sequence being transcribed (starts at start point)

Figure 14.9

Transcription factors

TATA box

Promoter Nontemplate strand

Start point

Transcription initiation complex forms.

Transcription initiation complex

DNA

RNA transcript

A eukaryotic promoter

Several transcription factors bind to DNA.

3ʹ 5ʹ

5ʹ 3ʹ 3ʹ 5ʹ

3ʹ 5ʹ

3ʹ 5ʹ

3

2

1 Template

strand

Transcription factors

RNA polymerase II

3ʹ 5ʹ

3ʹ 5ʹ T A T A A A A

A T A T T T T

In Eukaryotes transcription factors bind first and trigger the making of the primary RNA transcript (or pre-mRNA)

Figure 14.10

Nontemplate strand of DNA

Direction of transcription

RNA polymerase

3ʹ

5ʹ 3ʹ

5ʹ

RNA nucleotides

Newly made RNA

3ʹ end

5ʹ

U

U

G

A

A

A

A

A A

A

A

T T T

T T C C

C

C C C

G

U

10-20 nucleotides are exposed to make a transcription bubble

RNA polymerase adds nucleotides to make new primary RNA transcript.It is single stranded!

Note: RNA has Uracil instead of Thymine!

Figure 14.10

Nontemplate strand of DNA

Direction of transcription

RNA polymerase

3ʹ

5ʹ 3ʹ

5ʹ

RNA nucleotides

Template strand of DNA

Newly made RNA

3ʹ end

5ʹ

U

U

G

A

A

A

A

A A

A

A

T T T

T T C C

C

C C C

G

U

As the advancing wave of RNA synthesis takes place the new RNA molecule peels away from DNA template and helix reforms.

40 nucleotides per second! Uracil takes the

place of Thymine

A single gene may have multiple transcription points and multiple RNA polymerases working on it like ”trucks in a convoy”

What does that mean for the product (the protein that is being made)?

Figure 14.4

Nuclear envelope

Pre-mRNA

mRNA

DNA

RNA PROCESSING

TRANSCRIPTION

TRANSLATION

Polypeptide

Ribosome mRNA

DNA TRANSCRIPTION

TRANSLATION

Polypeptide

Ribosome

In Eukaryotes this initial RNA sequence is called pre-mRNA or the primary transcript or the primary RNA transcript.

It needs to go through “post-transcriptional modification or processing” before becoming a mature mRNA!

1. Ends modified (5’ cap, poly-A tail added)

(Why? Seems to help export process, may reduce degradation at ends, may help ribosome grab.)

Figure 14.12

31–104

5ʹ Cap

5ʹ UTR 3ʹ UTR

Poly-A tail

Coding segment

1–146

AAUAAA

105– 146

5ʹ Cap Poly-A tail 1–30

mRNA

Intron Intron

2. Interior sections or introns cut out and exons kept and spliced together by spliceosome.

This is called RNA splicing

So….the mRNA molecule that enters cytoplasm is a very abridged version!

Figure 14.12

Introns cut out and exons spliced together

31–104

5ʹ Cap Poly-A tail

Coding segment

1–146

AAUAAA

105– 146

5ʹ Cap Poly-A tail 1–30

mRNA

Intron Intron

RNA splicing is really amazing because…..

A single gene can encode more than one kind of polypeptide or protein –depends on which sections treated as exons!

So protein products are much more diverse than number of genes.

Figure 14.12

Introns cut out and exons spliced together

31–104

5ʹ Cap Poly-A tail

Coding segment

1–146

AAUAAA

105– 146

5ʹ Cap Poly-A tail 1–30

mRNA

Intron Intron

So lets say you were going to make a protein out of only two of the exons above…how many different proteins could you potentially make?

Figure 15.12

DNA

Primary RNA transcript

mRNA or

Exons

Troponin T gene

RNA splicing

1 2 3 4 5

1 2 3 5 1 2 4 5

1 2 3 4 5

Why is this great for the organism?

Figure 14.4

Nuclear envelope

Pre-mRNA

mRNA

DNA

RNA PROCESSING

TRANSCRIPTION

TRANSLATION

Polypeptide

Ribosome mRNA

DNA TRANSCRIPTION

TRANSLATION

Polypeptide

Ribosome

Where does mRNA go?

Figure 14.4b-3

Nuclear envelope

Pre-mRNA

mRNA

DNA

RNA PROCESSING

TRANSCRIPTION

TRANSLATION

Polypeptide

Ribosome

(b) Eukaryotic cell

While DNA stays safe and secure in nucleus, mRNA takes the chances, venturing out into the cells cytoplasm and mingles with a whole new crew of construction enzymes and protein-making factories called ribosomes.

Mature mRNA is exciting but what we really want is a protein!

To get a protein we need to make a polypeptide (a string of amino acids)

THIS IS TRANSLATION (happens at ribosomes)

Figure 14.5

DNA template strand

Protein

mRNA

3ʹ

Trp

TRANSCRIPTION

TRANSLATION

Amino acid

Codon

5ʹ

3ʹ 5ʹ

3ʹ

5ʹ

Phe Gly Ser

G U G U U U G G U C C A

C A C A A A C C A G G T

G T G T T T G G T C C A

NOTE…Uracil taking place of Thymine in mRNA!

Translation

mRNA heads out into cytoplasm to attach to ribosome

Figure 14.4b-3

Nuclear envelope

Pre-mRNA

mRNA

DNA

RNA PROCESSING

TRANSCRIPTION

TRANSLATION

Polypeptide

Ribosome

(b) Eukaryotic cell

Figure 14.17

P E A

tRNA molecules

A

Large subunit

Small subunit

Growing polypeptide Exit tunnel

E P

mRNA 5ʹ 3ʹ

Growing polypeptide (a) Computer model of functioning ribosome

tRNA

5ʹ

3ʹ E

mRNA

(c) Schematic model with mRNA and tRNA

Codons

Amino end Next amino acid to be added to

polypeptide chain

Large subunit

Small subunit

A site (Aminoacyl- tRNA binding site)

P site (Peptidyl-tRNA binding site)

Exit tunnel

E site (Exit site)

mRNA binding site

(b) Schematic model showing binding sites

What is a ribosome?? •  Are tons of these in cytoplasm •  Actually made up of a kind of RNA (ribosomal

RNA)•  Do you see mRNA above?

Figure 14.5

DNA template strand

Protein

mRNA

3ʹ

Trp

TRANSCRIPTION

TRANSLATION

Amino acid

Codon

5ʹ

3ʹ 5ʹ

3ʹ

5ʹ

Phe Gly Ser

G U G U U U G G U C C A

C A C A A A C C A G G T

G T G T T T G G T C C A

A focus on codons!Sets of three mRNA nucleotides are called codons. At ribosome each codon will match with a particular a.a.

Anther kind of RNA (transfer RNA or tRNA) is out in cytoplasm hanging around with amino acids!

tRNA has an anticodon at one end and hooks onto a specific amino acid at other end

if mRNA codon is GGCtRNA anticodon to match will be CCG

and would have grabbed glycine as its amino acid

CCG

GGC

Figure 14.14

5ʹ

tRNA

Polypeptide

Ribosome

Anticodon

mRNA

Codons 3ʹ

tRNA with amino acid attached

Amino acids

Gly

Trp

Phe

A A A

U U U G G C U G G

More Translation….

•  mRNA-gets conveyed thru the ribosome unit until the start codon (Start codon is always AUG )

•  Start codon establishes reading frame (every set of three after that is a codon)

•  Once hits start codon tRNA hauls appropriate amino acid to the ribosome

Figure 14.5

DNA template strand

Protein

mRNA

3ʹ

Trp

TRANSCRIPTION

TRANSLATION

Amino acid

Codon

5ʹ

3ʹ 5ʹ

3ʹ

5ʹ

Phe Gly Ser

G U G U U U G G U C C A

C A C A A A C C A G G T

G T G T T T G G T C C A

A little more detail about codons..

There are 64 possible codons. Why?

[4 possible bases (A, G, C, U) and 3 bases per codon so 43]

3 are stop codons! (and one start codon..so actual is 64-4=60)

BUT THERE are not 61 different amino acids Hmmmmm?

In reality there are multiple codons that match each amino acid.

mRNATable

What is wobble?

The idea that both AGA and AGG will both code for Arginine-3rd slot is more flexible

GGU,GGC,GGAwillalsomatchtoGlycine

Figure 14.17

P E A

tRNA molecules

A

Large subunit

Small subunit

Growing polypeptide Exit tunnel

E P

mRNA 5ʹ 3ʹ

Growing polypeptide (a) Computer model of functioning ribosome

tRNA

5ʹ

3ʹ E

mRNA

(c) Schematic model with mRNA and tRNA

Codons

Amino end Next amino acid to be added to

polypeptide chain

Large subunit

Small subunit

A site (Aminoacyl- tRNA binding site)

P site (Peptidyl-tRNA binding site)

Exit tunnel

E site (Exit site)

mRNA binding site

(b) Schematic model showing binding sites

Too much detail!

Terminology!

Polypeptide refers to a chain of amino acids… ��Protein is typically the finished product-how do you get that finished product?

FYI…it is also called a mature protein.

Post Translational Modifications p285Polypeptide starts to coil and fold due to its primary structure (its amino acid sequence) (might be a chaperone protein that helps it fold correctly)

•  Groups are added (sugars, lipids, phosphate groups)

•  Parts might be removed (e.g. amino acids from leading end or middle EX. Insulin is formed after a chunk of a.a. are taken out of its middle.)

•  Polypeptides may be joined together to become subunits of a big protein like hemoglobin

Post Translational Modifications

Once again we call the protein formed after these modifications a “mature” protein.

Prokaryotes do this but not as much as Eukaryotes…

Troubles with ends! (Not required material)During replication DNA polymerase can add only to 3’ end so cannot complete 5’end.

Repeated rounds of replication produce shorter and shorter DNA molecules with uneven ends. P259 txt

RNA primer replacement is not a problem at origins of replication within the chromosome, because DNA polymerase I can attach to an "upstream" piece of DNA to backfill where the RNA primer was. At the ends there is no 3’ piece of DNA available to use as a primer.

Figure 13.16

5ʹ 3ʹ

5ʹ

3ʹ

Origin of replication Lagging strand Lagging

strand

Overall directions of replication

Leading strand

Leading strand

Overview

Primase makes RNA primer.

RNA primer for fragment 1

Template strand

Okazaki fragment 1

DNA pol III makes Okazaki fragment 1.

DNA pol III detaches.

5ʹ 3ʹ

5ʹ

3ʹ

5ʹ

3ʹ 5ʹ

3ʹ

RNA primer for fragment 2

Okazaki fragment 2 DNA pol III

makes Okazaki fragment 2.

Overall direction of replication

DNA pol I replaces RNA with DNA.

DNA ligase forms bonds between DNA fragments.

5ʹ

3ʹ 5ʹ

3ʹ

5ʹ

3ʹ 5ʹ

3ʹ

5ʹ

3ʹ 5ʹ

3ʹ 1

2

3

4

5

6

(Not required material)Lets assume this is the end of the chromosome

1. RNA comes off….

2. DNA pol I is supposed to add by hooking onto 3’ piece of previous DNA stretch-oops

Qs for Telomere Article and p 258 text!1. What are telomeres? Do they contain genes? Do bacteria have telomeres?

2. What is telomerase and what does it do? What would happen in germ cells if telomerase did not exist?

3. Telomerase is not usually active in somatic cells, but turns on in germ cells, why?

4. Unusual activity of telomerase is often seen in what condition?

5. Non coding repetitive sequences?…Apoptosis? What do these things mean?

6. What kinds of conditions are associated with shortened telomeres?

7. What did the researchers find? Can you make a sketch of the findings the way our textbook does for experiments it describes?

8. Look at Table 1. What are the numbers in the parentheses after the means! 9. Look at Figure 1. What does the little star on the line between the two elderly groups mean?  

Figure1.TelomerelengthexpressedasT/Sra8oamongathletesandnon-athletes,stra8fiedbyage.

ØsthusIBØ,SguraA,BerardinelliF,AlsnesIV,etal.(2012)TelomereLengthandLong-TermEnduranceExercise:DoesExerciseTrainingAffectBiologicalAge?APilotStudy.PLoSONE7(12):e52769.doi:10.1371/journal.pone.0052769hZp://www.plosone.org/ar[cle/info:doi/10.1371/journal.pone.0052769

ErrorBars!Causeandeffect?Istelomerelengtharesultoftheirbeingathletes?OrAretheyathletesbecausetheyhavelongtelomeres?(Perhapslongtelomeresmaketheirbodies“work”beZer,recoverfromworkoutsfaster,meanstheyaremorelikelytobeathletes)

The diagram below shows a replication bubble with synthesis of the leading and lagging strands on both sides of the bubble. The parental DNA is shown in dark blue, the newly synthesized DNA is light blue, and the RNA primers associated with each strand are red. The origin of replication is indicated by the black dots on the parental strands.

Rank the primers (the red specks) in the order they were produced. If two primers were produced at the same time, overlap them.

The diagram below shows a replication bubble with synthesis of the leading and lagging strands on both sides of the bubble. The parental DNA is shown in dark blue, the newly synthesized DNA is light blue, and the RNA primers associated with each strand are red. The origin of replication is indicated by the black dots on the parental strands.

Rank the primers in the order they were produced. If two primers were produced at the same time, overlap them.a and h then b and g then c and f and finally e and d

True of Leading strand, Lagging strand, or Both????

Daughter strand elongates away from replication fork

Multiple primers needed

Made in segments

Made continuously

Daughter strand elongates toward replication fork

True of Leading strand, Lagging strand, or Both????

Daughter strand elongates away from replication fork Lag

Multiple primers needed Lag

Made in segments Lag

Made continuouslyLead

Daughter strand elongates toward replication fork Lead

In an analysis of the nucleotide composition of DNA, which of the following will be found? (Imagine counting number of nucleotides of each type)

A = G and C = T

G + C = T + A

A = C

A + C = G + T

In an analysis of the nucleotide composition of DNA, which of the following will be found?

A = G and C = T

G + C = T + A

A = C

A + C = G + T

Cytosine makes up 42% of the nucleotides in a sample of DNA from an organism. Approximately what percentage of the nucleotides in this sample will be thymine?

31%

42%

8%

16%

It cannot be determined from the information provided.

Cytosine makes up 42% of the nucleotides in a sample of DNA from an organism. Approximately what percentage of the nucleotides in this sample will be thymine?

31%

42%

8%

16%

It cannot be determined from the information provided.