Genetics Exam 2- Iii

Front 1

Characteristics of the Genetic Code

Back 1

1. Linear code of ribonucleotide letters
2. "words" are triplets of ribonucleotide
i.e. codons
3. Code is unambiguous
codon specifiies one aa
4. Code is degenerate
1 or more codons/ aa
5. Start and stop codons initiate and terminate translation
6. Translation without interruption (comma-less)
7. Non-overlapping code
8. Code is nearly universal

Front 2

How is genetic information encoded?

Back 2

mRNA

Front 3

mRNA

Back 3

unstable intermediate between DNA and the ribosomes that transfer the genetic information from DNA to proteins
mRNA is relatively unstable, especially when compared to DNA

Front 4

How are 20 aa's specified from 4 nucleotides?

Back 4

Brenner proposed that the genetic code existed as a triplet which would allow for 64 combinations of the 4 letters
Crick and colleagues found the frameshifts of 1 or 2 nucleotides caused mutations
Gain or loss of 3 nucleotides did not cause mutation.

Front 5

Deciphering the Genetic Code: Nirenberg

Back 5

Synthesized mRNAs using the enzyme Polynucleotide Phosphorylase
Used these artificial messages in an in vitro protein synthesis system to make polypeptide sequences.
Does not require DNA template- adds ribonucleotides randomly
Ribonucleotide that is added is dependent on the concentration of that ribonucleotide relative to the other three.

Front 6

Homopolymers

Back 6

Polymers of single nucleotides
e.g. polyU, polyA, polyG
Use homopolymers in in vitro translation and determine which amino acid was added to the newly synthesized protein
Used a mixture of the 20 amino acids, but only one was radioactively labeled so that it could be detected.

Front 7

RNA Heteropolymers (Copolymers)

Back 7

Add two or more different ribonucleotides to form the artificial mRNA.
Based on the ratio of ribonucleotides added, the composition of the codons can be predicted: Codon composition that is predicted to occur most frequently would be predicted to specify the amino acid that is found in the greatest amount in the peptide.
Allows the composition of the triplet codons to be deduced (although not the exact sequence of the codon)

Front 8

Triplet Binding Experiments to Determine Sequence of Codons

Back 8

Synthesized small tri-ribonucleotides
Ribosomes would bind these and cognate tRNA
Radioactively label single amino acids
Identify which amino-acid bound tRNA associated with the ribosome/tri-ribonucleotide complex
i.e. which labeled amino acid would be retained in the ribosome complex.

Front 9

Sequencing MS2 phage

Back 9

the DNA/RNA sequence and amino acid sequence of the encoded proteins were shown to be "co-linear"
5' end of the gene = NH terminal of the protein

Front 10

Universality of the Genetic Code

Back 10

MS2 phage
Cell free bacterial systems used to translate eukaryotic genes
Recombinant DNA molecules expressed correctly in different organisms.

Front 11

The Genetic Code

Back 11

Dictionary of 61 triplets (codons) for 20 amino acids
3 codons specify terminatino: UAA, UAG, and UGA
First two nucleotides in the codon more critical for attracting the correct tRNA to the mRNA.
3rd position = wobble position

Front 12

Stop Codons

Back 12

UAA, UAG, UGA

Front 13

Wobble position

Back 13

3rd position; has relaxed base pairing rules

Front 14

Translation start codon

Back 14

AUG (prokaryotes & eukaryotes)
rarely GUG in prokaryotes
Specifies N-formylmethionine (fmet)
formyl group removed after the protein is synthesized.

Front 15

Transcription

Back 15

Synthesis of RNA molecules form DNA template
RNA molecule is complementary to one strand of the DNA template
Triplet codons in the mRNA strand is complementary to an anticodon region of a specific tRNA molecule
mRNA transfers genetic information from DNA to protein.

Front 16

E. coli polymerase

Back 16

B a B' subunits provide the catalytic activity
a subunits provide scaffolding function and interact with other proteins
o factor provides a regulatory function
All RNA polymerases synthesize RNA in a 5'-3' direction

Front 17

O factor

Back 17

Different o factors provide specificity for binding of RNA pol to different promoters
o factor is not part of the holoenzyme.

Front 18

Bacterial Promoter Elements

Back 18

-35 and -10
- 35 : TTGACA
- 10: TATAAT (pribnow box = -10 region)

Front 19

3 stages of Transcription

Back 19

Initiation, Elongation, Termination

Front 20

Initiation

Back 20

Polymerase binds to the promoter sequence in duplex DNA (closed complex)
Polymerase melts duplex DNA near transcription start site, forming a transcription bubble (operon complex)
Polymerase catalyzes phosphodiester linkage of two initial rNTPs.

Front 21

Elongation

Back 21

Polymerase advances 3'-5' down template strand, melting duplex DNA and adding rNTPs to growing RNA.

Front 22

Termination

Back 22

At transcription stop site, polymerase releases completed RNA and dissociates from DNA.

Front 23

Eukaryotic Transcription

Back 23

Transcription and Translation occur in separate compartments: no coupling of transcription and translation
More DNA sequences and proteins involved in activating transcription
Eukaryotic mRNAs undergo post-transcriptional processing.

Front 24

Post-transcriptional processing

Back 24

addition of 5' cap structure and 3' poly-A tail
Removal of introns and splicing exons

Front 25

Introns

Back 25

intervening sequences (split genes)

Front 26

3 forms of RNA polymerase in Eukaryotic Cells

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I, II, and III.
I: rRNA
II: mRNA
III: tRNA

Front 27

Initiation of Eukaryotic Transcription

Back 27

cis-acting elements- DNA sequencing elements: serve as binding sites for trans-acting factors
e.g. TATA box, CAAT box, enhancers

Front 28

trans-acting factors

Back 28

transcription factors
bind cis-acting elements
Function to recruit RNA polymerase and promote transcription initiation

Front 29

General (Basal) Transcription Factors

Back 29

TFIID- complex that contains TATA box-binding protein (TBP)
Associate with the promoter at the TATA box and recruit RNA Pol II to form the Pre-initiation complex.

Front 30

Transcriptional Activators and Repressors

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Bind to enhancer elements to induce high levels of transcription or to repressor elements to repress transcription.
SP-1, NF-kB, CREB

Front 31

Steps in Transcription Initiation in Eukaryotes

Back 31

1. TFIID binds to the TATA box
2. Recruites other general factors and the RNA Pol II holoenzyme to form the PIC
3. Helicase activity of TFIIH unwinds the DNA at the promoter and allows RNA Pol II to initiate transcription
4. Once elongation begins, most of the general factors dissociate.

Front 32

Posttranscriptional Processing of RNA in Eukaryotes

Back 32

Pre-mRNA= heterogeneous RNA or RNA that has not been processed
RNA processing occurs in nucleus
As the new strand of RNA is being transcribed, a 5' cap is added
RNA processing at the 3' end of the transcript: poly-adenylation.
Splicing.

Front 33

3' Poly-adenylation

Back 33

a portion of the 3' end is cleaved and Poly-A Polymerase adds about 200 adenosine residues to the 3' end.
Poly A tail functions primarily in mRNA stability.