lecture 14 definitions

Nucleic Acid Function: Translation and Technologies

Transfer RNA (tRNA)

RNA “adapter” that carries amino acids one at a time to the ribosome for translation (protein synthesis).

Ribosomal RNA (rRNA)

RNA that makes up the ribosome, the catalyst (ribozyme) for protein synthesis.

tRNA is the ______ between the language of nucleic acids and the language of proteins.

“translator”

Amino acid loading of tRNA is driven by…

hydrolysis of PPi (2 ATP eq.)

Codon

Group of 3 bases of mRNA

Anticodon

Group of 3 bases of tRNA (reverse complement of mRNA)

The reading frame (how bases are put into groups) is determined by:

where in the sequence you start

The ability to transfer info stored as DNA to protein depends on…

complementarity of bases

Which codon would bind the following anticodon? 5’ CAU 3’

AUG

Translate the following gene: 5’ ATG CAG GTG TAA 3’

Met Gln Val Stp

Ribosome has 3 tRNA binding sites

• A site

• P site

• E site

A-site

amino acyl tRNA binding site

E-site

exit site, deacylated tRNA

P-site

peptidyl-tRNA binding site

translation rxn steps

1. protein chain attaches to tRNA at P-site

2. amino grp leaves to attack ester of tRNA-AA linkage in P-site

3. new amide bond formed

4. protein chain (n+1) is now attached to tRNA in A site

In which site of the ribosome is the peptide bond formed?

Between the A and P sites.

How many ATP equivalents are required for translation initiation?

1

steps of translation elongation

1. decode

2. form peptide (amide) bond

3. translocate (move one spot down)

how many ATP eq. for translation elongation?

2

How does the ribosome know it’s the correct tRNA?

It measures the size of the base pairs?

Why can there be a wobble at codon 3?

• Ribosome measures precisely at codon 1 and 2 for specific size of Watson-Crick base-pair, but can’t measure well at the 3rd so allows for wobble

steps of translation termination

1. codon in A site is a stop codon

2. release factors bind and allow water to hydrolyze ester linkage btwn protein and tRNA in P site

3. 1 GTP hydrolyzed to 1 GDP to separate pieces

How many ATP equivalents are required for translation termination?

1

Point mutation

change in a single base

Insertion

addition of one or more bases

Deletion

loss of one or more bases

Silent mutation

point mutation that does not change the amino acid (due to degeneracy in the genetic code)

Frameshift mutation

insertion or deletion that shifts the reading frame (happens if not multiple of 3; all sequence after mutation will be incorrect)

Single Nucleotide Polymorphism (point mutation)

• could cause disease or might not

• occurence in non coding regions of DNA could predispose or protect against disease

• the most common genetic variation among humans

Which point mutation is a silent mutation in the codon below? 5’ GAU 3’

5’ GAC 3’

Clones

Identical copies of a sequence of DNA (fragments or whole organisms)

in vitro means?

outside of cells

Minimum requirements for vitro DNA replication

• DNA polymerase

• DNA template

• 2 primers (1 each strand)

• dNTPS (building blocks)

Polymerase chain reaction (PCR)

• Fast and efficient way to make a lot of copies of DNA

(clones)

• Each “cycle” doubles the amount of DNA present (2n)

• Use DNA polymerase protein that is resistant to heat denaturation so that ds DNA can be denatured by just raising the temperature

How many times more DNA is present after 20 cycles of PCR?

1,000,000x

Recombinant DNA

• combo of DNA segments

• use bacteria as a host to make large amounts of DNA, RNA, or protein

Plasmid

circular DNA that can be propagated inside bacteria

Restriction endonuclease

Cut DNA asymmetrically at a specific sequence, leaving a single-stranded overhang

What is the complementary strand

for the HindIII restriction

endonuclease site, shown below?

5’ A//AGCTT 3

5’ A//AGCTT 3’

Viral genomes

Viruses can have either DNA genomes or RNA genomes, and these can be either single-stranded or double-stranded

• ds DNA (smallpox, adenovirus, herpes)

• ss RNA (coronavirus, HIV, hepatitis B, influenza)