Bio Exam 3 (2nd half)

Which molecule acts as the "translator" between nucleotide sequences and amino acids, and where is the amino acid specifically attached?

• Molecule: Transfer RNA (tRNA).

• Attachment Site: The 3' end (via a covalent bond). (amino acid covalent attached)

tRNAs are

single stranded RNAs but form a structure due to base pairing

Anti-codon in the tRNA (3’ to 5’) base pairs with

codon in mRNA (5’ to 3’) in the ribosome

What is the specific enzyme responsible for covalently attaching an amino acid to its corresponding tRNA, and what molecule provides the energy for this reaction?

• Enzyme: Aminoacyl-tRNA synthetase.

• Energy Source: ATP.

How does Aminoacyl-tRNA synthetase ensure the "genetic code" is accurately maintained during the attachment process?

The enzyme has a specific binding pocket that recognizes both the amino acid and the anticodon of the tRNA. This dual recognition ensures the correct amino acid is matched to the correct tRNA.

What is the functional significance of the high-energy bond created when an amino acid is attached to a tRNA?

The energy stored in this hihg energy bond is later used to drive the formation of the polypeptide chain (peptide bonds) inside the ribosome during translation.

mRNA is read

5’ to 3’ in sets of three nucleotides called codons

For every three bases in the mRNA there is

one amino acid incorporated into the growing polypeptide chain

There are 20 amino acids but only

4 nucleotides

4 bases x 4 bases x 4 base =

64 possible codons, 3 positions

The genetic code is

degenerate

degenerate

Multiple codons encode the same amino acid

A single tRNA can often recognize

more than one codon because of expanded base pairing: wobble

In most cases there is more than one tRNA for an

amino acid

What is the Wobble position, and what does it allow a single tRNA to do?

• Position: The 3rd nucleotide of the mRNA codon (which pairs with the 1st nucleotide of the tRNA anticodon).

• Function: It allows for non-standard base pairing (e.g., G pairing with U), meaning one tRNA can recognize and bind to more than one specific codon.

According to the slide, how do "Wobble" and "Multiple tRNAs" differ in how they handle the amino acid Leucine?

Wobble: The same tRNA anticodon can match two different codons.

More than one...: Different tRNAs with different anticodons can still carry the same amino acid (Leucine).

The ribosome is the

cell’s decoder

The ribosome is made of

proteins and RNA and is a large complex

What are the two primary components of a bacterial ribosome shown in the structural model?

1. Large ribosomal subunit (the top, light green section).

2. Small ribosomal subunit (the bottom, dark green section).

If we strip away the protein in a bacterial ribosome,

Ribosomal RNAs give the ribosome its overall shape

Small Ribosomal Subunit function

Matches tRNA to mRNA (decoding).

Large ribosomal subunit function

catalyzes peptide bond formation (linking amino acids)

RIbosome sites

Exit site, Pass site, Add site

When translation begins sets the “reading frame” it must be

precise

IN eukaryotes, initatior tRNA with

Methionine binds with the P-site of small subunit with translation initation factors

In eukaryotes, the small ribosomal subunit (with bound initiation factors) recognizes the

5’ cap on the mRNA and scans until it finds the first AUG (codes for Met)

Shine-Dalgarno sequence

Prokaryotic translation initiation begins with this special sequence in mRNA

Specific sequences in the mRNA base pair with the RNA in the small subunit of the ribsome,

perfectly positioning the initiatior formyl-Methionine (f-met) tRNA over the start codon AUG, setting the reading frame

Protein production occurs in a

4 step cycle (same process for both eukaryotic and prokaryotic cells)

4 step cycle of protein production

• Step 1: tRNA Binding – A newly bound charged tRNA enters the A site by base-pairing with the mRNA codon.

• Step 2: Peptide Bond Formation – The polypeptide chain is uncoupled from the tRNA in the P site and joined by a peptide bond to the amino acid linked to the tRNA in the A site. (This is catalyzed by the large subunit).

• Step 3: Large Subunit Translocation – The large subunit moves forward relative to the mRNA, shifting the spent tRNA into the E site and the peptidyl-tRNA into the P site.

• Step 4: Small Subunit Translocation – The small subunit moves forward 3 nucleotides, ejecting the "empty" tRNA from the E site. The A site is now empty and ready for the next charged tRNA.

Why must where translation ends be precise?

Mutations that alter a stop codon are called read-through mutations

Release factor (protein not tRNA)

catalyzes hydrolysis reaction which release polypeptide chain from the P-site tRNA (same for both eukaryotic and prokaryotic)

Dissociated ribosomal subunits can bind a new

mRNA

Polysome/Polyribosome

Many ribosomes can bindone mRNA

Scientists can measure the number of ribosomes on a mRNA to get a sense of

the efficiency of translation

How to decode an mRNA

• Identify Cell Type: Determine if the mRNA is eukaryotic or prokaryotic.

• Orient Direction: Read the mRNA in the 5' to 3' direction.

• Find Start: Locate the AUG (Met) codon to set the reading frame.

• Read Triplets: Group nucleotides into codons (triplets) and translate using the codon table.

• Terminate: Stop decoding once you reach a Stop codon (UAA, UAG, or UGA).

What is the likely phenotypic outcome if 1 or 2 nucleotides are added/deleted in the DNA compared to adding/deleting 3 nucleotides?

• 1 or 2 Nucleotides: Causes a frameshift mutation, which alters every subsequent codon in the sequence, usually resulting in a completely different or non-functional protein.

• 3 Nucleotides: Results in the addition or loss of a single amino acid but preserves the rest of the reading frame.

Eukaryotic mRNAs are

monocistronic

monocistronic

one gene, one mRNA, one protein/polypeptide chain

Unlike in prokaryotes, the ribosome cannot enter the mRNA anywhere except by

scanning from 5’ cap into the mRNA

Many bacterial mRNAs are

polycistronic

Polycistronic

one mRNA, more than one polypeptide chain/protein

The ribosome in bacterial mRNAs can initiate translation from anywhere in the transcript that has a

Shine-Delgarno sequence followed by an AUG start codon

In alternative codon table read from the

center out

DNA mutation can lead to

changes in mutation

Silent mutation

DNA change results in the same amino acid (due to the redundancy of the genetic code). No change to protein function.

Nonsense mutation

DNA change results in a premature STOP codon. This truncates the protein, usually making it non-functional.

Missense mutation

DNA change results in a different amino acid.

Base pair insertions and deletions cause

frameshifts

Since all living organisms use DNA we can perform experiments using

recombinant DNA technologies, harnessing the power of the central dogma

What are some uses of recombinant DNA/molecular cloning/ genetic engineering?

• Protein expression & purification for biochemical analysis 

• Expression of tagged proteins (for localization studies) 

• Generation of mutant proteins (for functional analysis) 

• Creation of proteins with new functions (mixing and matching protein domains to create proteins with new functions)

• Generation of bacterial strains allows for making lots of copies of DNA; used for sequencing, long-term storage, transfer to another organism (ie yeast, mouse or human cells)

We want to express a human gene in a

bacterial cell

All living organisms use the

Central dogma to make proteins (excluding some proteins)

Recombinant DNA allowed us to express human insuling in bacteria instead of harvesting it

from pig pancreas

Compare and contrast transcription in prokaryotes vs. eukaryotes.

Similarities

• Both use DNA to make mRNA for protein synthesis

• Both have 3 stages of transcription:

  1. Initiation

  2. Elongation

  3. Termination

• RNA polymerase synthesizes RNA in the 5’ → 3’ direction

• RNA polymerase reads the template strand 3’ → 5’

• Transcription starts at the +1 site

• Promoters determine where transcription begins

• Multiple RNA polymerases can transcribe a gene at the same time

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Prokaryotic Transcription

• Only one RNA polymerase

• Sigma factor helps RNA polymerase bind promoter

• Promoter contains -35 and -10 regions

• No introns

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Eukaryotic Transcription

• Three RNA polymerases

  • RNA Pol II makes mRNA

• Uses TBP + general transcription factors

• Promoter often contains a TATA box

• RNA Pol II has a CTD tail important for regulation

• Introns are present and must be removed by splicing

What must be modified to express a human gene in bacteria?

Promoter Differences

• Bacteria use sigma factor + RNA polymerase to initiate transcription

• Human promoters will not work in bacteria

• A bacterial promoter with -35 and -10 elements must be added

• This promoter is usually provided by the plasmid vector

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Intron Differences

• Eukaryotic genes contain introns

• Bacteria cannot splice introns out

• Scientists use cDNA (made from processed mRNA) instead of genomic DNA

• cDNA contains only exons, allowing bacteria to make the correct protein

Why do scientists use cDNA instead of genomic DNA when expressing human genes in bacteria?

• Human genomic DNA contains introns

• Bacteria lack the machinery to splice out introns

• cDNA is made from processed mRNA and contains only exons

• Using cDNA allows bacteria to produce the correct protein

How does the poly-A tail help in making cDNA?

• Eukaryotic mRNA has a poly-A tail

• A poly-T primer binds to the poly-A tail

• This provides a starting point for reverse transcriptase

• Reverse transcriptase then synthesizes DNA from the mRNA template

What enzyme makes DNA from an RNA template during cDNA synthesis?

• Reverse transcriptase

• Uses mRNA as the template

• Produces a complementary DNA strand (cDNA)

• Commonly derived from retroviruses

What are the steps of cDNA synthesis?

1. Isolate mature mRNA

2. Bind poly-T primer to poly-A tail

3. Reverse transcriptase synthesizes first DNA strand

4. RNase degrades RNA

5. DNA polymerase synthesizes second DNA strand

6. Double-stranded cDNA is produced

What is the final product of cDNA synthesis?

• Double-stranded complementary DNA (cDNA)

• Contains only coding sequences (exons)

• Lacks introns

• Can be inserted into plasmids for bacterial expression

What do we need to replicate DNA? (PCR)

1. Template sequence (use our cDNA)

2. A way to separate the double stranded template DNA. Heat takes the place of helicase

3. Primers ○ We use synthesized DNA primers, so we eliminate the need for primase, nuclease, and ligase

4. Enzymes – DNA polymerase

5. Nucleotides

Bacteria need to:

• Copy the plasmid

• Promote expression (transcription/translation) of the inserted gene

• Allow for insertion of new genes

• Retain the plasmid

Plasmid engineered with:

• Origin of replication (ori)

• -35/-10 promoter to recruit RNA polymerase

• Shine-Dalgarno sequence

• Multiple cloning site (MCS)

• Selectable marker

What is a plasmid vector?

A small circular DNA molecule used to carry and replicate foreign DNA inside a host cell, usually bacteria.

Why must we consider differences between prokaryotes and eukaryotes?

The plasmid is placed in bacteria, so gene expression depends on bacterial proteins and machinery, even if the gene encodes a human protein.

What are promoter elements in a bacterial plasmid?

DNA sequences (such as the -35 and -10 regions) that allow bacterial RNA polymerase to start transcription.

What is the Shine-Dalgarno sequence?

A bacterial ribosome-binding site that helps initiate translation of mRNA into protein.

What is a Multiple Cloning Site (MCS)?

A short DNA region containing multiple restriction enzyme sites used for inserting target genes.

Why is maintaining the reading frame important during insertion?

An incorrect reading frame can change codons and produce a nonfunctional protein.

What is the origin of replication (ori)?

A DNA sequence that allows the plasmid to replicate inside the host cell.

Why is the origin of replication important?

It enables production of many copies of the plasmid within bacteria.

What is a selectable marker?

A gene, often antibiotic resistance, used to identify bacteria that contain the plasmid.

What does the selectable marker allow researchers to do?

Distinguish transformed bacteria (with plasmid) from non-transformed bacteria.

What role do restriction enzymes like EcoRI play in cloning?

They cut DNA at specific sequences to allow insertion of DNA fragments into the plasmid.

What is a recombinant plasmid?

A plasmid that contains inserted foreign DNA

What organism is commonly transformed with recombinant plasmids?

Escherichia coli (E. coli).

What is the overall purpose of a plasmid vector?

To clone, replicate, and express a target gene inside a host organism.

Why is a selectable marker (like an antibiotic resistance gene) included in a plasmid when transforming bacteria?

• The Problem: Most bacteria in a sample do not successfully take up the plasmid.

• The Solution: The marker allows only the transformed cells to survive. When the sample is spread on a plate containing the drug (antibiotic), the non-transformed cells die, and only those expressing the resistance gene survive to form colonies.

In the provided diagram of the selection process, what does the presence of only two red colonies on the final plate indicate?

It indicates that only two individual bacterial cells successfully took up the plasmid and expressed the drug resistance gene, allowing them to grow into colonies while the rest of the sample was killed by the drug in the agar.

Recombinant DNA Steps

1. PCR sequence of interest containing proper restriction enzyme sites at the 5’ and 3’ end (using cDNA as template) 2. DIGEST the backbone and insert with the same restriction enzyme(s) 3. LIGATE the insert with the vector in a test tube (outside of the cell) 4. TRANSFORM into bacteria cells 5. PLATE onto petri plates with agar and drug to kill any cells that did not take up plasmid 6. SCREEN for colonies with insert, isolate the plasmid and confirm by restriction fragment mapping 7. USE cells containing proper plasmid for your experimen

Expression plasmids contain

a promoter element, multiple cloning site, origin of replication, and selectable marker

Digestion with restriction enzymes and DNA gel electrophoresis is used to

map the plasmid and confirm gene insertion

In Agarose Gel Electrophoresis, what determines the direction and speed of DNA movement?

• Direction: DNA is negatively charged and moves toward the positive electrode.

• Speed: Shorter DNA fragments move faster and further through the gel matrix than longer DNA fragments.