Protein synthesis

1
Protein synthesis

• Transcription Translation

2
Why does Protein Synthesis have to happen the way
it does?

• Where is DNA stored?
• Inside the nucleus
• Where are proteins made?
• In the cytoplasm on ribosomes
• So how do we get outside the nucleus to the
  cytoplasm?

DNA
Nuclear envelope
3
Why cant DNA leave?

• Lets brainstorm some problems with this...
• DNA too valuable
• If it were damaged it would be useless
• Proteins are required in large amounts
• By only having two identical DNA strands it will
  take too long
• Once used by the ribosome, DNA has to re-enter
• Lots of potential problems here

4
So what is a cell to do?

• Use messenger RNA!!!
• DNA is transcribed into a complementary RNA
  message
• Multiple copies can be made and delivered to
  ribosomes in the cytoplasm
• Ribosomes then translate the message to make a
  polypeptide chain
• Therefore, protein synthesis divided into two
  processes
• Transcription ? creating an RNA strand
• Translation ? amino acids assemble based on RNA

5
RNA

• Recall...

6
How RNA differs form DNA

• RNA

• DNA

• RNA has a sugar ribose
• RNA contains the base uracil (U)
• RNA molecule is single-stranded

• DNA has a sugar deoxyribose
• DNA has thymine (T)
• DNA is double-stranded

7
Structure of RNA
8
Types of RNA

• Messenger RNA (mRNA) carries genetic information
  to the ribosomes
• Ribosomal RNA (rRNA), along with protein, makes
  up the ribosomes
• Transfer RNA (tRNA) transfers amino acids to the
  ribosomes where proteins are synthesized

9
Making a Protein

• The basics...

10
Genes and Proteins

• Proteins are made of amino acids linked together
  by peptide bonds
• 20 different amino acids exist
• Amino acid chains are called polypeptides
• Segment of DNA that codes for the amino acid
  sequence in a protein are called genes

11
Protein Synthesis Overview

• Transcription makes an RNA molecule complementary
  to a portion of DNA
• Initiation, elongation, termination
• Involves RNA polymerase, promoter region,
  terminator sequence
• Translation occurs when the sequence of bases of
  mRNA DIRECTS the sequence of amino acids in a
  polypeptide
• Initiation, elongation, termination
• Involves ribosomes, tRNA, start codon, stop codon

12
Genetic Code

• DNA contains a triplet code
• Every three bases on DNA stands for ONE amino
  acid
• Why 3 bases?
• There are 20 amino acids with a possible 64
  different triplets
• If one nucleotide coded for one a.a., we would
  have 4
• If two nucleotides coded for one a.a., we would
  have 16 (42 16)
• Three nucleotides (43) 64 nucleotides plenty!
• Each three-letter unit on mRNA is called a codon
• The code is nearly universal among living
  organisms

13
Genetic Code

• Most amino acids have more than one codon!
• Why?
• Redundancy reduces possible errors that may lead
  to serious mutations
• Start Codon ? AUG (most of the time)
• Stop Codon(s) ? UGA, UAA, UAG

14
Genetic Code
15
Protein Synthesis Overview
16
Homework

• Page 241 1-6, 8, 10-11
• Shall we play codon bingo??!!

17
Transcription

• Initiation
• Elongation
• Termination
• and some fixing...

18
Initiation

• RNA polymerase binds to DNA to be transcribed and
  opens up the double helix
• RNA polymerase binds to a specific site upstream
  from the gene to be transcribed ? promoter region
• Called TATA box
• Made of a string of thymine and adenines
• Indicates where the RNA polymerase should start
  transcribing and which DNA strand to transcribe

19
Initiation
20
Initiation

• Why TATA?
• of bonds between A-T 2
• Easier to break 2 bonds to get the process going
• Therefore requires less energy from RNA polymerase

21
Elongation

• RNA polymerase has bonded to promoter, opened up
  DNA and starts building mRNA strand
• Travels in 5 3 direction
• No primer required (unlike DNA replication)
• Promoter region is not transcribed
• RNA polymerase only transcribes one strand
• Template strand
• Strand not used for transcription
• Coding strand

22
Elongation

• mRNA strand complementary to template strand and
  identical to coding strand

23
Termination

• RNA polymerase continues synthesizing mRNA until
  it reaches terminator sequence
• New mRNA strand separates from DNA template
• Transcription ceases
• RNA polymerase able to find another promoter
  region and transcribe another gene

24
Termination
25
Posttranscriptional Modifications

• Primary transcript ? mRNA that needs to be
  modified before it can leave nucleus
• Modifications to be made...
• Capping
• Tailing
• Removal of introns

26
Posttranscriptional Modifications

• Capping
• 5 cap added to start of primary transcript
• Made of 7-methyl guanosine
• A modified guanine nucleoside triphosphate
• Function
• Protects mRNA from digestion in the cytoplasm
• Helps it bind to a ribosome as part of initiation
  of translation

5 cap
mRNA
27
Capping
28
Posttranscriptional Modifications

• Tailing
• A string of approximately 200 adenine
  ribonucleotides added to 3 end of mRNA strand
• Called poly-A tail
• Added by poly-A polymerase

mRNA
Poly-A polymerase
Poly-A tail
29
Tailing
30
Posttranscriptional Modifications

• Introns and Exons
• Exons ? coding regions of DNA
• Introns ? non-coding regions of DNA (like genetic
  gibberish)
• Introns must be removed
• Why?
• If these non-coding regions are translated, the
  protein wont fold properly
• Therefore, dysfunctional protein

31
Posttranscriptional Modifications

• Spliceosomes
• Particles of RNA and protein that cut introns
  from mRNA primary transcript AND join exons
  together
• Coding region now continuous
• Spliced out introns stay behind in nucleus and
  are degraded

32
Posttranscriptional Modifications
33
Moving out

• Primary transcript now capped, tailed and introns
  excised
• Transcript now called mRNA transcript
• Ready to leave nucleus, attach to ribosome and
  begin translation!

34

• Note
• No quality control enzymes to make sure mRNA
  molecule is accurate (like DNA replication)
• More errors made during transcription
• But its okay...since many mRNA copies made, good
  odds that most will be correct and well have
  enough functional proteins

35
Homework

• Page 249 1-5, 8

36
Translation

• The RNAs
• The Ribosome
• Initiation, Elongation, Termination

37
Translation Overview

• Synthesis of proteins in the cytoplasm
• Involves the following
• 1. mRNA (codons)
• 2. tRNA (anticodons)
• 3. ribosomes
• 4. amino acids

38
Messenger RNA (mRNA)

• Carries the information for a specific protein
• Made up of 500 to 1000 nucleotides long
• Sequence of 3 bases called codon
• AUG methionine or start codon
• UAA, UAG, or UGA stop codons

39
Messenger RNA (mRNA)
40
Transfer RNA (tRNA)

• Made up of 75 to 80 nucleotides long single
  stranded
• Role ? Transports amino acids to the mRNA
• At one end, picks up the appropriate amino acid
  floating in the cytoplasm
• At the other end, has anticodons that are
  complementary to mRNA codons
• Recognizes the appropriate codons on the mRNA and
  bonds to them with H-bonds
• E.g. if mRNA codon is AUA, anticodon is UAU and
  carries tyrosine

41
tRNA
Once corresponding amino acid has attached to its
tRNA acceptor site at the 3 end, it is called
aminoacyl-tRNA Facilitated by aminoacyl-tRNA
synthesases At least 20 of these enzymes one
for each particular amino acid and tRNA
42
tRNA The wobble hypothesis

• The third base in each codon may differ between
  two codons that code for the same amino acid
• E.g. UAU and UAC both code for tyrosine
• The tRNA with anticodon AUA can still bond to UAC
• Flexibility helps with possible errors made
  during mRNA synthesis

43
The Ribosome

• Made of a large and small subunit
• Composed of rRNA (40) and proteins (60)
• Bind to mRNA at 5 cap
• Initiation of translation
• Have two sites for tRNA attachment
• P ? peptide site
• A ? acceptor site

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The Ribosome

Large subunit
P Site
A Site
Small subunit
45
The Ribosome

• Ribosomes move along mRNA in 5 3 direction
• Amino acid is added each time a ribosome reads a
  codon

46
The Ribosome

• Reading frame
• The phase in which to read the mRNA as groups of
  three
• Possible problem
• If ribosome doesnt start reading at the
  appropriate nucleotide, it could affect the
  polypeptide sequence
• E.g. AUGCCAGAU... can have the following reading
  frames
• AUG CCA GAU... or UGC CAG AU...
• It depends on where the ribosome begins reading
• Therefore, it is important that the mRNA be
  positioned properly

47
Translation

• Three steps
• 1. initiation start codon (AUG)
• 2. elongation amino acids linked
• 3. termination stop codon (UAG, UAA, or UGA).

Lets Make a Protein !
48
Initiation

• Start codon (AUG) is recognized by ribosome
• Ensures correct reading frame
• AUG codes for methionine
• tRNA carrying methionine enters P site (official
  initiation)

49
mRNA Codons Join the Ribosome

Large subunit
P Site
A Site
Small subunit
50
Initiation

anticodon
A
U
G
C
U
A
C
U
U
C
G
A
hydrogen bonds
codon
mRNA
51
Elongation

• The next tRNA carrying the appropriate amino acid
  enters the A site
• First amino acid bonds to second amino acid
• Ribosome shifts (translocates) one codon
• tRNA in P site leaves and is recycled
• Now tRNA that was attached to A site is moved to
  P site
• 3rd amino acid enters the now available A site
  and bonds to 2nd amino acid via peptide bond
• Process continues until stop codon is reached

52
Elongation
peptide bond
aa1
aa2

1-tRNA
2-tRNA
anticodon
U
A
C
G
A
U
A
U
G
C
U
A
C
U
U
C
G
A
hydrogen bonds
codon
mRNA
53
aa1
peptide bond
aa2

1-tRNA
U
A
C
(leaves)
2-tRNA
G
A
U
A
U
G
C
U
A
C
U
U
C
G
A
mRNA
Ribosomes move over one codon
54
peptide bonds
aa1
aa2
aa3
2-tRNA
3-tRNA
G
A
U
G
A
A
A
U
G
C
U
A
C
U
U
C
G
A
A
C
U
mRNA
55
peptide bonds
aa1
aa2
aa3
2-tRNA
G
A
U
(leaves)
3-tRNA
G
A
A
A
U
G
C
U
A
C
U
U
C
G
A
A
C
U
mRNA
Ribosomes move over one codon
56
peptide bonds
aa1
aa2
aa4
aa3
3-tRNA
4-tRNA
G
A
A
G
C
U
G
C
U
A
C
U
U
C
G
A
A
C
U
mRNA
57
peptide bonds
aa1
aa2
aa3
aa4
3-tRNA
G
A
A
4-tRNA
G
C
U
G
C
U
A
C
U
U
C
G
A
A
C
U
mRNA
Ribosomes move over one codon
58
Termination

• Ribosome reaches stop codon
• UGA, UAG, UAA
• Dont code for a.a., so no incoming tRNAs
  process stops
• Release factor
• Recognizes process stopping
• Aids in release of polypeptide chain from
  ribosome
• Two ribosome subunits fall off mRNA translation
  officially finished
• Primary structure of protein is made

59
aa5
aa4
Termination
aa199
aa200
primary structure of a protein
aa3
aa2
aa1
terminator or stop codon
200-tRNA
A
C
A
U
G
U
U
U
A
G
C
U
mRNA
60
Homework

• Page 254 1a-c, 2-4, 6, 7, 9