Biol 190 Lec 19

What is a gene?

What is a gene?

Sequence of nucleotides within a chromosome that provides the instructions for RNA synthesis and eventually a functional protein; macromolecule: nucleic acid

What is a genome?

All of the genes within the body

What is a DNA template strand?

One of the separated parental strands (AKA OG strand); 3' to 5'

What is the process of gene expression?

Define gene expression.

The process by which information encoded in DNA directs the synthesis of proteins

Can one gene make more than one polypeptide?

Yes, but polypeptides of the same family

Compare gene expression in bacterial cell versus eukaryotic cell.

-Bacteria: DNA transcribed in cytoplasm->mRNA is translated into polypeptide (in cytoplasm)

-Eukaryote: DNA transcribed in nucleus-> Pre-mRNA undergoes RNA processing-> mRNA comes out of the nucleus into cytoplasm-> ribosomes will convert mRNA into a polypeptide (translation)

What are the three differences of RNA from DNA?

Uracil, single-stranded, ribose sugar

Does pre-mRNA (primary RNA) exist in prokaryotes?

No

Why is DNA not used directly/ why is RNA used?

1. Protection: there is only one copy of each gene in the entire cell

2. Amplification: transcription of one gene can make many RNAs

3. Control: adds another level of control over expression of protein product

What are the results and location for pre-mRNA "processing"?

Results: mRNA (mature RNA); Location: Leaves nucleus through nuclear pore

What are the results and location of translation?

Results: Proteins; Location: Cytoplasm

What is the similarity between replication and gene expression?

Both use DNA template strands

What is the difference between replication and gene expression?

Replication: turns DNA into more DNA; Gene expression: -DNA->mRNA; only one strand of the DNA template is used (template strand)

What is the template strand not used in gene expression called?

Non-template (coding) strand

What is messenger RNA (mRNA)?

the RNA copy of a gene used in the cell to produce a polypeptide (protein); same sequence of the non-template strand except T is replaced by U; antiparallel

____% of DNA...

99; does not make proteins

____% of DNA is genes

1

What is the promoter region?

A sequence before a gene (sequence upstream of a gene); signifies the presence of a gene

Does the promoter region become part of a protein?

No

What are the two roles of the promoter?

1. Positions RNA polymerase

2. Identifies template strand (promoter reads the template strand)

What happens at a promoter?

Transcription factors (proteins) sit at a promoter (every gene has its own combo of transcription factors); correct combo of transcription factors allows RNA polymerase to sit on the promoter

What are the three stages of transcription?

1. Initiation

2. Elongation

3. Termination

What occurs at the first stage of transcription?

Initiation:

-requires the right combination of transcription factors on promoter

-RNA polymerase binds the promoter

What does RNA polymerase do?

1. Begins unwinding the DNA strands

2. Begins synthesizing mRNA at the start point

Does RNA polymerase require a primer?

No, by itself it can catalyze the formation of RNA using a template

What occurs at the second stage of transcription?

Elongation:

-only one mRNA strand is made by RNA polymerase

-DNA template strand is read and complementary mRNA is made from a 5' to 3' direction

-RNA polymerase rewinds the DNA behind it

Does the first and second stage of transcription create a lagging strand?

No

What occurs at the third stage of transcription?

Termination: RNA polymerase "falls off" the template strand where the RNA polymerase/DNA interaction is unstable (where a signal signifies the end of the gene)

What are the termination signals called for bacteria and eukaryotes?

-Bacteria: termination signal

-Eukaryotes: polyadenylation signal (AAUAAA)

What must occur before eukaryotic RNA leaves the nucleus?

RNA processing:

-modification of mRNA ends

-RNA splicing

Does one gene equate to one protein?

No, RNA splicing makes one gene equal more than one protein

What does the alteration of mRNA ends entail?

-5' cap: 5' end receives a modified nucleotide (guanine)

-poly-A tail on the 3' end: a lot of adenines on 3' end

What are the functions of alternating the mRNA?

-Aid the export of mRNA to cytoplasm

-Protect mRNA from degradation

-Help ribosomes attach to the 5' end during translation

What would happen if the alterations did not prevent degradation?

DNAses (enzymes like DNA) & RNAses would live and destroy single-stranded nucleic acids

What alternating segments do pre-mRNA and DNA genes have in common with?

Coding and noncoding segments

What are introns?

Noncoding regions (does not provide instructions for proteins)

What are exons?

Expressed, translated sequences (coding regions)

What is the purpose of RNA splicing?

Removes introns and joins exons, creating an mRNA molecule with a continuous coding sequence

How does RNA splicing cut introns out?

A snRNP (small nuclear ribonucleoprotein) at each junction generates a spliceosome (complex of RNA and enzymatic proteins) from accessory factors to join two exons together by splicing out introns

What happens to the introns after being cut out?

Consumed by DNAses to become nucleotides again

Why do introns exist?

An intron for one mRNA can be an exon for another mRNA

Define alternative splicing.

Exons from the same gene are joined in different combinations, leading to different mRNA transcripts; creates protein diversity

Can the same gene make different protein isoforms?

Yes, the different exons of one gene can form different domains in a new protein

What is the main structural formation of a protein?

Discrete regions called domains

What happens if an exon is not used?

Spliced out-> the absence of an entire (internal) region from a protein

What makes RNA so versatile?

1. RNA is single-strand-> form hydrogen bonds with its bases to DNA, proteins or other RNAs

2. Forms a 3D structure because of its ability to base pair with itself (w/ hydrogen bonds)

3. Acts as an enzyme (ribozymes) b/c of its varied structure and functional groups

What are ribozymes?

An RNA enzyme; RNA catalyst (i.e. proteins are not the only catalyst)

Are RNAs self-splicing?

Some are

What are the four players that direct protein synthesis?

1. mRNA

2. Amino acids

3. tRNA

4. Ribosome

Describe the first player in direct protein synthesis.

mRNA: the "blueprint" intermediate; every three RNA nucleotides represent one codon

Define codons.

Three nucleotides that codes for one amino acid in a polypeptide (not considered a protein yet); 3 nucleotides-> 1 codon-> 1 amino acid

Are codons in the polypeptide?

No

Describe the second player in direct protein synthesis.

Amino acid: multiple codons can encode the same amino acid (redundancy); no ambiguity (e.g. UCU is Serine and cannot represent another amino acid)

What is the 3rd position of a codon/base called?

Wobble: the only position that can change without making a different amino acid

How many start codons (beginning codon that starts a polypeptide) are there? Name them.

1; Methionine (AUG)

How many stop codons (termination signals) are there?

3

How many amino acids are there?

20

Describe the third player in direct protein synthesis.

tRNA: a 4 cloverleaf (arms) structure with codon/anticodon interactions are held together by H+ bonds; function: carry amino acid needed for translation; an amino acid covalently binds to its 3' end by the enzyme: aminoacyl-tRNA synthetase

How many aminoacyl-tRNA synthetases are there?

20 for each of the 20 amino acids

What is the structure of an aminoacyl-tRNA synthetase?

Two parts in its active site:

1. For amino acids

2. For tRNA; needs ATP to bind the amino acid to tRNA

What happens when an amino acid binds to a tRNA?

tRNA becomes charged (due to the charged carboxyl group of the amino acid attaching to the tRNA)

Describe the interaction between the anticodon (tRNA) and the codon (mRNA).

Complementary bases form hydrogen bonds between the antiparallel anticodon (tRNA: 3'->5') and codon (mRNA: 5'->3') stabilizing the interaction

The ribosome knows which amino acid to add to the growing polypeptide chain by the ___________ interaction.

Codon/anticodon

What is the coding strand and what is the template strand between anticodons and codons?

Coding strand: codon; Template strand: anticodon

Describe the fourth player in direct protein synthesis.

Ribosome: composed of two rRNA (small and large) subunits and proteins; site of translation where mRNA, tRNA, and rRNA are present

Describe the large subunit of the ribosome.

Has three locations: E, P, A

-A site: first site; aminoacyl-tRNA binding site (charged tRNA sits here)

-P site: peptidyl-tRNA binding site; a different tRNA w/ a growing polypeptide chain linked to it

-E site: exit site; a third tRNA w/ nothing on it

Describe the small subunit of the ribosome.

Has the mRNA binding site

What does accurate translation require?

A correct match between a tRNA and an amino acid (mRNA codon), done by the enzyme aminoacyl-tRNA synthetase

What energy is used for translation?

GTP

What are the three steps of translation?

1. Initiation

2. Elongation

3. Termination

What occurs at the first step of translation?

Initiation:

1. The small ribosomal submit binds the mRNA first (at the cap) and scans along until it finds the start codon, AUG (codes for methionine attached to the initiator tRNA)

2. The large ribosomal subunit attaches to begin translation (requires energy)

What components are involved in the first step of translation?

1. tRNA + methionine

2. Small ribosomal subunit

3. Both bound to mRNA

What occurs at the second step of translation?

Elongation: Large subunit catalyzes 2 reactions:

1. Breaks the bond between the growing polypeptide chain and the tRNA (at the P site)

2. Catalyzes formation of peptide bond between Met and the next aa; every aa needs 2 GTP

What occurs at the third step of translation?

Termination:

1. Ribosome reaches a stop codon on mRNA

2. Release factor (protein) promotes hydrolysis

3. All components dissociate

How does the polypeptide become a protein?

1. The first few amino acids are a signal peptide that tell the cell where to go with the polypeptide

2. Signal-recognition particle (SRP) binds to the signal peptide to take it to the lumen of the Rough ER

3. SRP binds to receptor protein at the Rough ER

4. SRP detaches, synthesis resumes

5. Enzyme cuts off signal peptide

6. Polypeptide folds into final confirmation

What are post-translational modifications that can occur to a mature polypeptide?

Enzyme assistants:

1. Proteolysis (polypeptide cuts into thirds, each piece acts as a protein)

2. Phosphorylation

3. Glycosylation (adds sugar)

-all takes place in the golgi apparatus

What is the coupling of transcription and translation?

Overlapping of the two processes/occur at the same time

Can prokaryotes and eukaryotes couple transcription and translation?

P: Yes, both occurs in cytoplasm; E: No, transcription (nucleus) and translation (cytoplasm) occur in different places

Why can bacteria grow so fast?

Can couple transcription and translation (multiple ribosomes can translate a single mRNA simultaneously, forming a polyribosome); do not need to process mRNA