Lecture 13-14 / Chapter 12 - The Genetic Code and Transcription

What are the eight characteristics of the genetic code?

1. (Nearly) Universal

2. Linear

3. In Frame

4. Unambiguous

5. Degenerate

6. Punctuated

7. Commaless

8. Non-overlapping

Mnem: Understand, lewd indecency under dictator policing could never.

“The genetic code is (nearly) universal?”

The same universal genetic dictionary is used by all organisms with some exceptions.

“The genetic code is linear?”

The genetic code is written in linear form, using the ribonucleotide bases that compose mRNA molecules as letters.

“The genetic code is in frame?”

Each ‘word’ within the mRNA consists of three ribonucleotide letters, producing a triple code.

With several exceptions, each group of three ribonucleotides constitutes a codon, which specifies one amino acid.

“The genetic code is unambiguous?”

Each triplet specifies only a single amino acid.

(Codon → one amino acid)

“The genetic code is degenerate?”

A given amino acid can be specified by more than one triplet codon

(Amino acid → several codons)

“The genetic code is punctuated?”

The code contains one “start” and three “stop signals, triplets that initiate and terminate translation.

“The genetic code is commaless?”

No internal punctuation, that is no pausing and restarting until the protein is synthesized.

“The genetic code is non-overlapping?”

Once translation commences, any single ribonucleotide at a specific location within the mRNA is part of only one triplet.

mRNA?

An ____ molecule transcribed from ____and translated into the ____sequence of a ____.

It’s also known as the ____.

An RNA molecule transcribed from DNA and translated into the amino acid sequence of a polypeptide.

It’s also known as the unstable intermediate template.

What direct evidence do we have that information in DNA is not translated directly?

• An unstable intermediate template was observed and evidence of its importance accumulated.

How is it possible that only four letters could specify 20 words?

Provide the mathematical evidence.

Given only four letters, different permutations would then provide the variety needed to specify the codons of the 20 amino acids in the universal genetic code.

4¹ = 4

4² = 16

4³ = 64 > 20, so a codon length of 3 is the minimum needed to encode for all 20 amino acids.

How is it possible that only four letters could specify 20 words?

Name the experiment that provides evidence to this.

Crick et al. studies of the Reading Frame

What is the premise of the Crick et al. reading frame experiments?

We observe that certain indels lead to frameshifts while others don’t

By using indels of various lengths, we can determine the reading frame of the DNA.

Results of the Crick et al. reading frame experiments?

• When in/del one nucleotide, a frameshift mutation occurred.

• When in/del two nucleotides, a frameshift mutation occurred.

• When in/del three nucleotides, the reading frame was reestablished.

  • When in/del multiples of three, the reading frame is reestablished.

List the five things that are necessary for protein synthesis in vitro:

1. Ribosomes

2. tRNAs

3. Amino Acids

4. Translation factors

5. Polynucleotide phosphorylase

What activity does PNPase perform in bacterial cells?

It degrades RNA in bacterial cells.

What activity does PNPase perform in vitro?

It builds RNA.

How are we able to make PNPase perform the opposite reaction in vitro?

We must introduce a high concentration of rNDP.

What principle difference is there between RNA pol and PNPase?

What happens to the ribonucleotide sequence as a result?

RNA pol requires a template. PNPase doesn’t.

The sequence ends up being random based on the relative concentration of the four rNDPs added.

In an in vitro PNPase experiment, how is the probability of a specific ribonucleotide added related to the availability of each rNDP?

\propto

List the order of the experiments that helped us determine the universal genetic code.

Earliest

1. Homopolymer Experiments (Nirenberg-Matthaei)

2. Heteropolymer Experiments (Nirenberg-Matthaei-Ochoa)

3. Triplet Binding Assay Experiments (Nirenberg-Leder)

4. Copolymer Experiments (Khorana)

Latest

Homopolymer?

mRNA homopolymer?

A polymer consisting of only one kind of monomer?

All U, all A, all C, or all G.

Procedure of the Homopolymer Experiments?

• Label 1/20 possible amino acids

• Translate 1/4 possible homopolymers

  • If the resulting proteins are radioactive, then the correct amino acid was used.

Which three codons were assigned through the Homopolymer Experiments?

UUU = phenylalanine

AAA = lysine

CCC = proline

Which codon was not able to be assigned through the Heteropolymer Experiments?

Why is this?

GGG. The homopolymer would fold in on itself, preventing translation.

What is the premise of the Heteropolymer Experiments?

If you know the relative proportion of each type of rNDP, you could predict the frequency of any particular triplet codon occurring in the synthetic mRNA.

Frequency of each amino acid corresponds to the frequency of triplet(s). ← Recall that the code is degenerate.

Why were 1/6 and 5/6 the proportions used in the Heteropolymer Experiments?

Really, as long as the proportions aren’t 1:1, the experiment will still work.

These two values were chosen because they gave distinct enough values when summed and multiplied.

What is the minimum length of an mRNA’s nucleotide sequence for a ribosome to be able to bind to it?

Just three nucleotides

tRNA?

A small ribonucleic acid molecule with an essential role in translation.

“Walk around” the clover structure of tRNA starting from the 5’-end.

5’-end

• 5’-pG end of the acceptor stem

• D loop

• Anticodon loop

  • Anticodon

    • Wobble position

    • Middle position

    • Distal position

• Variable loop

• TψCG loop

• CpCpA-OH-3’ end of the acceptor stem

3’-end

Anticodn?

Three-base segment that recognizes a codon in mRNA

Binding site?

A site for the specific amino acid corresponding to the anticodon.

Recognition sites?

Sites for interaction with ribosomes and with the enzyme that links the tRNA to its specific amino acid.

How can we tell if a tRNA is attached to an amino acid?

The tRNA will gain a charge and the tRNA-amino acid complex would be larger in size than both individual molecules.

What is the procedure of the Triple Binding Assay Experiments?

• Different amino acids are added

• Labeled tRNAs with certain anticodons are also included and tagged with radiation.

• Everything is passed through a nitrocellulose filter.

  • We then check to see if the filter has radioactive substances on it.

What predictions were made for the Triple Binding Assay Experiments?

• If the amino acid being tested corresponds to the radioactive tRNA, then a radioactive aminoacyl-tRNA complex will form that will collect on the filter.

• If the amino acid being tested doesn’t correspond to the radioactive tRNA, then no complex has formed and the filter will not test positive for radiation.

What were the two major conclusions of the Triple Binding Assay Experiments?

1. The code is degenerate.

2. The code is unambiguous.

Copolymer?

Long RNA molecules consisting of short sequences repeated many times

What was Khorana’s procedure for making copolymers?

1. Khorana created short but identical sequences (di-, tri-, tetranucleotides)

2. He then joined them together using PNPase in vitro to produce the copolymer.

What did Khorana’s Copolymer Experiments discover?

The specific sequences of the codons in the DNA.

Khorana saw that certain copolymers led to truncated proteins. What is the explanation for this?

Stop codons. UAA UAG UGA.

Genetic coding dictionary?

The chart that allows you translate codon → amino acid.

How do you read a genetic codon dictionary?

1st letter - left side

2nd letter - top side

3rd letter - right side

If you look for degeneracy in the genetic codon dictionary, what will you observe?

Patterns of degeneracy - Codons that differ by only one letter will code for the same amino acid.

What observation of the genetic codon dictionary is the basis for the Wobble Hypothesis?

Pattern of degeneracy.

Wobble Hypothesis?

Crick’s proposal that the third base in an anticodon in tRNA that can align in several ways to allow it to recognize more than one base in the codons of mRNA.

What chemistry allows for wobble?

The hydrogen bonding at the third position of the codon-anticodon interaction is less constraned, so it doesn’t adhere as specifically to established base-pairing rules.

What does the 5'-end of the mRNA correspond to on the tRNA?

The 3’-end of the tRNA.

5’-end of tRNA (1st position) can have five possible codons. What are they?

A, C, G, U, I

What do the 1st position tRNA nucleotides correspond to in the 3rd position mRNA nucleotides?

A - U

C - G

G - C, U

U - A, G

I - A, U, C

“cug agu auci”

Why does wobble exist?

It minimizes or even removes the damage caused by substitution mutations in the third mRNA codon.

Why do tRNAs have their unique base pairing?

The less strict base pairing means that more codons can be accounted for with less tRNAs. It’s an economy measure.

What pattern appears when varying just the second letter in the codon?

The amino acids will be different but they’ll have similar chemical properties. Integration of the incorrect amino acid will not affect protein function as much. This once again minimizing the damage done by a substitution mutation.

Bacteriophage MS2?

A retrovirus bacteriophage that infects E. coli—its nucleic acid contains only about 3500 ribonucleotides, making up only four genes.

What is the virus that Fiers et al. worked with?

Bacteriophage MS2.

What was the goal of Fiers et al.’s work with MS2?

To sequence the four genes of the MS2 and their products.

What are the results of Fiers et al.’s work with MS2?

1. The RNA and the protein exhibit colinearity.

2. There is punctuation (start and stop).

What evidence do we have for the universality of DNA?

1. Translation occurs despite the origin of the actors (origin of mRNA, translation factors, etc.)

2. The same dictionary is observed in both bacteria and eukaryotes.

3. Synthesis of insulin in E. coli.

mtDNA

Double-stranded, self replicating circular DNA found in the mitochondria that encodes mitochondrial ribosomal RNAs, transfer RNAs, and proteins used in oxidative respiratory functions of the organelle.

What evidence do we have against the universality of DNA?

Mitochondrial DNA (observed in both yeast and humans) have different codes than usual. UGA codes for TRP instead of stop.

How do observations of translation of mtDNA provide evidence towards the wobble hypothesis?

The differences in the code are variations of the third position of the codon.

Why do we see the differences in dictionary between most DNA and mtDNA?

It shows a trend towards needing less tRNAs in mitochondria.

Overlapping genes?

The idea that one mRNA can have multiple initiation points for translation and thus one gene can produce various gene products.

How do overlapping genes not violate the nonoverlapping nature of DNA?

Each letter still only belongs to one word, but the word may be different depending on the initiation point for translation (and thus the reading frame)

How is φX174 unusual? Why does this happen?

Despite its small DNA sequence, it can produce many more gene products than one would expect (11 vs. 6).

This must be a result of multiple initiation.

Name an advantage that comes from overlapping genes.

The use of overlapping reading frames optimizes the use of a limited amount of DNA present in these small viruses.

Name a disadvantage that comes from overlapping genes.

A single mutation may affect more than one protein. The mutation has a much higher chance of being deleterious.

What evidence suggests that RNA is the intermediate between DNA and protein?

1. DNA and protein synthesis appear in different domains (nucleus vs. cytoplasm)

2. RNA is synthesized in the nucleus of eukaryotic cells where DNA is found.

3. RNA is chemically similar to DNA.

4. Most mRNA migrates out of the nucleus into the cytoplasm following synthesis.

RNA Polymerase?

An ____ that catalyzes the formation of an ____ using the ____ sequence of a ____ as a template.

An enzyme that catalyzes the formation of an RNA polynucleotide strand using the base sequence of a DNA molecule as a template.

Recall: Core enzyme?

The subunits of an enzyme necessary for catalytic activity.

What are the subunits of the core enzyme of bacterial RNA Pol?

• 2 copies of the α subunit

• β and β′

• ω

What additional enzyme needs to be added to turn the core enzyme of bacterial RNA Pol into the holoenzyme?

σ

Which subunits of the bacterial RNA Pol core enzyme provide the catalytic basis and active site for transcription?

β and β′

In summary, what role does σ play in RNA Pol?

It plays a regulatory function in the initiation of RNA transcription.

What is the difference between the template strand and the coding strand?

Template: The strand that is transcribed by RNA pol during transcription.

Coding: The strand named so because it is similar in 5’-3’ sequence to the RNA transcript (except T→U)

Upstream? Downstream?

Upstream: toward the 5’-end of the template strand, or before the transcription start site.

Downstream: toward the 3’-end of the template strand, or after the transcription start site.

cis-acting? trans-acting?

Cis-acting: DNA sequence that regulates the expression of a gene located on the same chromosome.

Trans-acting: A gene product (usually a diffusible protein or RNA molecule) that aacts to regulate the expression of a target gene.

What are the three (or maybe four) steps of Transcription?

1. Initiation (May include template binding, or it could be separate.)

2. Elongation

3. Termination

In bacterial DNA, what trans-acting factor interacts with what cis-acting factor to do template binding?

The σ factor of the bacterial RNA Pol.

Consensus sequence?

The sequence of nucleotides in DNA or amino acids in proteins most often present in a particular gene or protein.

This gives us a ‘best guess’ of what the most typical sequence looks like for a given segment of DNA or protein.

What cis-acting factor are consensus sequences a part of?

Promoters.

Promoter?

Region where RNA pol binds and initiates transcription.

Where is the promoter located within the gene?

The promoter is located upstream of the coding sequence.

t/f: The transcription start site is immediately after the promoter sequence.

False.

What number is the transcription start site designated?

+1

What two consensus sequences have been found in bacterial promoters?

-10 5’-TATAAT-3’

-35 5’-TTGACA-3’

What is the name of promoter -10 5’-TATAAT-3’?

Pribnow box.

Promoters are _-acting DNA elements.

cis-acting.

What two kinds of promoters are found in promoters?

Strong, weak.

How does sequence variation in promoters contribute to the variable gene expression?

Sequence variation → Difference in binding ‘strength’ → Variable gene expression.

Strong promoter?

Promoter where initiation can occur every 1-2 seconds.

Weak promoter?

Promoter where initiation can occur every 10-20 minutes.

How can a mutation in promoter sequence affect gene expression?

If the promoter sequence is mutated, it’s possible that it can no longer be identified by the RNA pol.

→ severe reduction of gene expression

What is the major form of the σ subunit in bacterial RNA Pol?

σ⁷⁰.

What does variation of σ factor in RNA pol allow for?

Difference in what genes are regulated, where promoters with different σ subunits recognize different promoter sequences.

Initiation

When the RNA Pol inserts the first 5’-rNTP.

1. t/f: A primer is required for initiation of RNA synthesis.

2. With regards to the answer to part 1, RNA synthesis is unlike what process when it comes to the need for a primer?

1. False.

2. DNA synthesis, DNA pol requires a primer.

Elongation?

The continuation of RNA polymerization when ribonucleotide complements are inserted and linked by phosphodiester bonds.

What direction does RNA synthesis occur in?

Downstream; 5’ to 3’.