D1.2

Define and summarize transcription

• Def: process where genetic information encoded in DNA is copied into RNA

• produces mRNA

• occurs in nucleus (eukaryotes) or cytoplasm (prokaryotes)

• Mainly uses RNA polymerase

What are the 3 steps in transcription

1. Initiation

• RNA polymerase binds to DNA at the beginning of a gene (at promoter region, regulated by transcription factors)

• Separates DNA strands by breaking H-bonds

2. Elongation

• RNA polymerase builds mRNA on a DNA strand (the template/antisense strand)

  • It moves along DNA reading one base at a time and adding free RNA nucleotides

3. Termination

• a terminator sequence in DNA is reached and mRNA is released

• RNA polymerase detaches from DNA, so both strands come together

What is the sense vs. antisense strand?

Sense strand = coding strand

Antisense strand = template strand

How is the complementary base pairing rule seen in transcription?

• Allows the correct RNA nucleotide to be placed by RNA polymerase

• It temporarily forms H-bonds

How is transcription related for gene expression?

• Transcription enables cells to switch on or off genes, depending on the products that are needed

Define gene expression

• the process where genetic info encoded in genes are used to synthesize proteins

Define translation and summarize it

• Def: the code from mRNA is read by ribosome and used to synthesize polypeptides

• Occurs in cytoplasm and in ribosomes

Describe the 3 components in translation

1. mRNA

• brings code from DNA

2. Ribosomes

• site of translation

• brings mRNA and tRNA in correct orientation

• has large subunit (where 2 tRNA binds) and small subunit (mRNA binds)

3. TRNA

• single stranded RNA that folds to create clover shape

• has double-stranded regions and 3 hairpin loops

• each tRNA has a specific AA attached

• when it recognizes and binds to code on mRNA on ribosome, tRNA transfers the appropriate AA to make polypeptide

What enables correct AAs to be put int he correct sequence to produce a functional polypeptide?

• The complementary base pairing rule

• codons are on mRNA, whereas anticodons are on tRNA

  • they are complementary to each other

What does the degeneracy of the genetic code mean?

• It means redundancy

• means that some AA are coded for by more than 1 codon

What are the 3 general steps in translation? Explain the process of elongation

• initiation, elongation, termination

• Begins with a new tRNA binding at the A site

  • tRNA has a specific AA that matches the second codon on mRNA

• the large ribosomal subunit catalyzes a reaction to make a peptide bond between the new AA and the AA held by previous tRNA in P site

• Ribosome translates along mRNA, one codon at a time

• the tRNA that was in P site moves to E site and exits

• tRNA in A site moves to P site, making A site available

• essentially, it allows the formation of a polypeptide chain

• since the ribosomal subunits hold mRNA and tRNA together so AA can be connected by peptide bonds

What is a point mutation and what are its different types?

• A single nucleotide is changed

• deletion, addition, substitution

• can occur during replication and transcription

What types of mutations are most dangerous/significant? why?

• deletion and insertion

• because it causes a frameshift mutation, where all codons are altered

What effect would a substitution mutation have?

• Depends

• due to degeneracy of genetic code, it might produce the exact same AA (silent mutation) —> usually if the third base is altered

• If the first or second base is altered, it will likely lead to different AA

What is an example of a disease related to a point mutation?

Sickle cell anaemia

Changes a gene producing haemoglobin —> dramatically changes the shape of haemoglobin

Which direction does mRNA move through a ribosome?

5’ to 3’ direction

Where can control be exerted in gene expression?

• The initiation of transcription

• The promoter (region of code on DNA before a gene) allows transcription factors to bind

  • The binding of the correct transcription factors and orientation allows RNA polymerase to bind and begin transcribing DNA into RNA

What happens if transcription factors are missing or can’t bind?

• Transcription won’t take place, the gene can’t be expressed

List 4 noncoding regions of DNA and its purpose

1. Regulators of gene expression (promoters)

2. Introns: noncoding DNA sequences in eukaryotic genes that are transcribed into RNA but removed during post-transcriptional processing

3. Telomeres: repetitive sequences protecting end of chromosomes

• ensures DNA is replicated correctly

• each time cell division occurs, small amounts of DNA are lost from telomeres

4. Genes to make rRNA and tRNA

What are introns vs. exons?

Introns: non-coding genes but has controlling sequences to regulate gene transcription

Exons: DNA sequences that code for polypeptides

found in eukaryotic genes

What are the 3 steps in modifying RNA for translation?

1. transcription

2. Adding a 5’ cap and a poly-A tail (protects mRNA from degradation)

3. Splicing (removing introns and joining exons to make mature mRNA) using spliceosomes

What is alternative splicing? Give an example

• when a gene is spliced in multiple ways by combining different exons and omitting others

• to create different proteins

• ex. in a foetus, troponin T gene is sliced in one way to be more sensitive to low blood pH —> after birth, its spliced in a different way and loses these properties

Describe the initiation stage of translation

• Mature mRNA (5’ terminal) binds to small ribosomal subunit on the binding site

  • Ribosome moves along mRNA until finds AUG

  • the anticodon of initiator tRNA (methionine) binds to codon on mRNA

  • large ribosomal subunit joins to finish the translation complex

• translation starts

Where are polypeptides modified to become functional?

• Carried by vesicles to the Golgi apparatus

Describe the post-translational modification of insulin

1. the direct product from translating insulin gene is pre-proinsulin. It has 4 sections (a signal peptide, A chain, B chain, C-peptide)

2. Pre-proinsulin enters the rough ER, where the signal peptide is removed. Now, its called proinsulin.

3. Disulfide bridges form between A and B chain

4. Proinsulin is packaged into vesicles that move to the Golgi apparatus

5. C-peptide is removed and mature insulin remains

Define the proteome

the total of all proteins made and used by the body

What do proteasomes do? what are they?

• They are protein complexes

• helps recycle and hydrolyse proteins to reuse amino acids

• Allows cells to maintain a supply of amino acids and getting rid of non functional ones

Describe the 3 major types of RNA in translation

• All are single stranded and are transcribed from a gene

• Note: subunits of ribosomes are made of rRNA molecules and proteins

How does mRNA get out of the cytoplasm for translation?

• Moves out through nuclear pores

List the general 9 steps in translation

1. mRNA binds with small ribosomal subunit

2. tRNA molecule with a specific AA attaches to the large subunit of the ribosome (P site). it combines with mRNA through complementary base pairing

3. A second tRNA with AA follows the first tRNA, base pairs with mRNA, and attaches to large subunit (A site). Two tRNAs can bind simultaneously to large subunit

4. An enzyme catalyzes a reaction between the 2 AA, forming a peptide bond

5. The first tRNA breaks free of its AA, detaches (move to E site), and floats into the cytoplasm (where it can get a new AA)

6. ribosome moves down mRNA molecule

7. the second tRNA is in the position that the first tRNA occupied

8. a third tRNA floats and is in the next sequence of bases on mRNA

9. Another peptide forms. continues until polypeptide is assembled

Describe the codons on tRNA

• they are in the middle loop

• they determine which one of the 20 AA is attached

Which direction is transcription written? What about translation?

• Its assembled 5’ to 3’

• the 5’ ends of free RNA nucleotides are added to the 3’ end of the RNA molecule being synthesized

• in translation, mRNA is read in 5’ to 3’

What determines which DNA strand will be the template strand?

• The promoter region

What are 2 regions needed for transcription?

1. promoter (binding site for RNA polymerase)

2. terminator

How does initiation of transcription differ in bacterial vs. eukaryotic cells

bacterial

• after RNA polymerase attached to promoter region

eukaryotic

• transcription factors bind to the promoter region of a gene first then attract RNA polymerase to initiate transcription

• DNA opens and a transcription bubble forms

What are transcription factors?

• proteins that play a role in regulating transcription process

• can attach directly to promoter or away from it

• determines which genes are active in cells

• allow attachment of RNA polymerase to promoter region so transcription begin (eukaryote)

  • More transcription factors can come later

Can transcription factors activate and prevent transcription?

Yes

What is involved to make a functional mRNA

• The first RNA formed is called pre-mRNA, which has exons and introns

• to make a functional mRNA, introns are removed through splicing

• during splicing, introns are removed, exons might be “rearranged” and a cap and polyA tail is added

what are spliceosomes?

• small nuclear RNAs and proteins

• help remove introns from primary mRNA

Describe the 3 sites on ribosomes. Where does decoding of mRNA occur?

• in the space between the 2 subunits of ribosome

• there are 3 sites for binding of tRNA and other sites for mRNA

What are the 5 steps in initiation in translation?

1. Initiation complex forms. involves an initiator tRNA pairing with mRNA start codon. Small ribosome subunit attaches to 5’ terminal of mRNA

2. the large subunit of ribosome is added

3. when complete ribosome formed, initiator tRNA is bound to P site and A site is empty

4. tRNA carrying the next AA enters ribosome A site.

5. after initiation, translation proceeds with elongation and termination

Where is the start codon found

At the 5’ end of all mRNA

How do eukaryotic cells mark the unneeded or damaged proteins

Use a chemical called ubiquitin

List the functions of ribosomes in translation

• translation: process of reading mRNA to form AA/peptides/proteins

• small subunit attaches to mRNA

• large subunit attaches to tRNA

• ribosomes move 1 codon at a time, in 5’ to 3’, to read mRNA

• catalyzes formation of peptide bonds

differentiate transcription vs translation

b. transcription produces RNA AND translation produces polypeptide/protein

c. RNA polymerase used in only in transcription and ribosomes only in translation

d. transcription in the nucleus «of eukaryotes» and translation in the cytoplasm

e. tRNA needed for translation but not transcription

f. nucleotides linked in transcription and amino acids in translation

   OR

   sugar-phosphate/phosphodiester bonds in transcription and peptide bonds in translation

what are tandem repeats for DNA profiling

Tandem repeats are short, repeating DNA sequences found in non-coding regions. The number of repeats varies widely between individuals and is inherited. In DNA profiling, these repeat regions are amplified and separated by gel electrophoresis or similar techniques. Each person has a distinct pattern of repeat lengths, making them useful for identity testing, paternity analysis, and forensic matching.