Chapter 15 - Transcription & Translation

Used the ch. 15 wrap-up and slides

Order of events for the central dogma

DNA → RNA → Protein

1. Replication on DNA inside the nucleus

2. DNA is transcribed into RNA inside the nucleus

3. RNA is translated into proteins in the cytoplasm

4. Protein synthesis occurs in ribosomes in the cytoplasm

Beadle and Tatum’s experiment

One enzyme, one gene hypothesis - surmised that each gene would influence a specific step in a metabolic pathway

Knowledge formed based on their findings:

1. Genes encode for proteins other than enzymes

2. Genes may encode a subunit

3. Some genes encode for non-coding RNA’s

4. Many genes have more than one exon & can be processed differently to develop different proteins

Define the term gene

specific sequence of nucleotides on a strand of DNA

• aid i the development of specific traits

• usually result in a protein product

• before protein synthesis… an RNA copy called “transcript,” of the gene must be made (mRNA)

Reading Genetic Code 

A gene is interpreted by reading the codons but… these codons must be read in the correct reading frame

• Which nucleotide starts the first codon of the coding region of the gene

• Always starts with the “start codon” AUG which encodes the amino acid Methionine

Prokaryotic gene expression

1. Happens solely in the cytoplasm

2. Do not require RNA transcript modification- their RNA transcripts can be translated immediately after being transcribed

3. RNA transcript can be transcribed and translated at the same time

• multiple polymerases can transcribe a single gene

• numerous ribosomes can concurrently translate the mRNA transcripts into polypeptides (polyribosomes)

  • This can allow a specific transcript and/or specific protein to rapidly reach high concentration in a cell

Eukaryotic gene expression

transcription and mRNA modification occur in the nucleus

translation occurs in the cytoplasm

Overall process of transcription

The gene sequence determines the sequence of bases along the length of a mRNA molecule. RNA is compromised of A, U, C, G

Occurs in the nucleus of eukaryotic cells

RNA polymerase is THE enzyme (she is THAT girl)

What do promoter sequences do in the initiation phase of transcription?

A sequence of DNA that serves as recognition and recruitment site for transcription factors and RNA polymerase

What do transcription factors do in the initiation phase of transcription?

Proteins that aid in the initiation and regulation of transcription

What do RNA Polymerases do in the initiation phase of transcription?

Synthesize the RNA transcript

Breaks hydrogen bonds between DNA strands and links together RNA nucleotides (same base pairing rules except RNA uses uracil instead of thymine)

RNA polymerase can only add nucleotides onto the 3’ end of the growing RNA transcript

Several types (I, II, III)

What are the steps for initiation in transcription

1. Transcription factors bind to promoter region of the gene needing transcription

2. T-factors recruit RNA polymerase to bind and form an initiation complex

3. RNAP will then recognize this start sequence and begin to synthesize the RNA transcript in a 5’ to 3’ direction

• Reads 3’ to 5’

• Writes 5’ to 3’

What is the importance of the elongation phase in transcription

It is where the production of the RNA transcript occurs

What is the role of RNAP in the elongation phase in transcription

Unwinds DNA to access the template strand (only exposes around 10-20 nucleotides at a time)

Connects the RNA nucleotides using DNA as a template

• produces RNA transcript in a 5’ to 3’ direction

• Temporary attachment

What are the steps for the elongation phase of transcription

1. RNAP unwinds DNA

2. RNAP reads 3’ to 5’ direction the the DNA nucleotides from the template strand and attaches RNA nucleotides

3. RNA nucleotides are joined to the previous one on the 3’ end by a phosphodiester bond along its backbone

What is the role of terminator sequences in the termination phase of transcription

sequences of DNA at the end of a gene that is transcribed and signals the RNA transcript is complete

What are the steps for termination in transcription

1. RNAP reaches and transcribes the termination sequence 

2. RNA transcript is released by RNAP

3. RNAP detaches from the DNA, ending transcription

GOAL of termination phase is to completely disassociate RNAP from DNA by stopping transcription and releasing the new RNA strand

How does the termination phase of transcription vary for prokaryotes versus eukaryotes

Pro- RNAP reads through a termination sequence and causes it to dissociate from the DNA, RNA is immediately ready for translation

Euk- RNAP reads through Polyadenylation sequence, the end of RNA transcript is then bound by proteins causing the RNAP to disassociate from DNA, RNA will need additional processing

What are the two mechanisms put on the ends of mRNA transcript

5’ cap and the Poly-A-tail

What is the function of the 5’ cap

modified guanine nucleotide placed on the 5’ end of the mRNA transcript

Necessary to maintain mRNA stability and recognition

What is the function of the poly-A-tail

placed on the 3’ end of mRNA transcript, string of 50-250 Adenine nucleotides that can vary in number depending on the transcript, cell type, and organism

Tail acts as a timer for how long you stay in the cytoplasm as a functioning mRNA

How does RNA splicing work and why is it important

Process of removing introns and joining together exon sequences to form a mature mRNA transcript

• Ensures that only coding sequences are translated; exons

• Removes introns (noncoding sequences) and joins exons

spliceosomes - complexes made of protein and catalytic RNA (riboenzymes)

Why do we care about introns

They allow for alternative splicing - the inclusion of differing sets of exons or differing mature mRNA produced from the same gene

Polypeptides within proteins often have discrete structural and functional regions called domains, each exon can encode for a different domain

Generally speaking, what happens during translation

The mRNA sequence determines the sequence of amino acids in the primary structure of the polypeptide; RNA-directed synthesis of a polypeptide

What is tRNAs role in translation

1. carry a specific amino acid on 1 end

2. have an anticodon on 1 end

3. single RNA strand that is about 80 nucleotides long

4. utilize a specific Aminoacyl-tRNA synthetase to attach its amino acid 

What is the ribosomes role in translation

Protein and rRNA complex that facilitates the reading of mRNA and production of the corresponding polypeptide

• achieved through the pairing of mRNA codons with tRNA anticodons

• Consists of 2 ribosomal subunits: large which brings in tRNA, and small that binds mRNA

• Has 3 binding sites: E, P, and A

What is the role of mRNA in translation

Molecule that directs the recruitment of tRNA molecules and production of the polypeptide

• Very specific sequence of RNA, unique to the polypeptide it will be used to create

mRNA is bonded to the small ribosomal subunit

• read every 3 base codons in a 5’ to 3’ fashion

• AUG = start codon

Each codon is bonded to a tRNA anticodon

What is the role of the polypeptide in translation

End product of translation by the assembly of amino acids bonded together in a specific sequence 

• interaction of one tRNA in the P site with another tRNA in the A site

• Directed by the bonding of mRNA codon to the anticodon of a tRNA

• Occurs in the ribosomes found in the cytoplasm of the cell 

What are the steps of the initiation phase in translation

Assembly step that brings mRNA, initiator tRNA, and 2 subunits of the ribosome together

1. mRNA binds the small ribosomal subunit

2. Start codon is located

3. Initiator tRNA binds to start codon

4. Energy is used to recruit and bind large ribosomal subunit

What are the steps of the elongation phase in translation

Reading stage where amino acids are bonded to each other; building a polypeptide chain out of the P site

1. tRNA enters the A site and bonds its anticodon with the corresponding codon of mRNA 

2. The growing polypeptide chain is attached to tRNA in P site, forms a peptide bond with amino acid attached to the tRNA in the A site

3. mRNA is shifted further along, causing bonded tRNAs to shift sites. tRNA in the P site breaks its bond with mRNA and enters E site. The tRNA in the A site is shifted to the P site as it now has the growing polypeptide chain attached

What are the steps of the termination phase in translation

Stage is reached when the stop codon is recognized in the mRNA 

1. Stop codon in the mRNA is reached and recognized 

2. Release factor is recruited and binds to the stop codon causing the hydrolysis of the polypeptide from the tRNA

3. Bonding + energy is utilized to cause the disassociation of the translation components

Point mutations

changes in a single base pair within the genome, when occuring in a gene these mutations can affect the structure and function of the overall gene product

Substitution point mutation

Silent (synonymous) - the single base pair change does not cause an amino acid change in the polypeptide

Missense (non-synonymous) - the single base pair change causes an amino acid change in the polypeptide; can be detrimental or not noticed at all

Nonsense - the single base pair change causes a change from an amino acid to a stop codon; very detrimental

Insertion point mutation

the addition of one base pair within the genome

Deletion point mutation

the removal of one base pair within the genome

Frameshift mutations

changes in nucleotide base pairs within the genome that causes a reading frame shift

Can occur as insertion or deletion

Chromosomal mutations

Very large scale nucleotide changes within a chromosome’s DNA

Insertions - addition of many nucleotides into the chromosome

Deletions - removal of a section of the chromosome

Translocations - part of one chromosome is removed and inserted to somewhere in another chromosome

Inversions - reversal of a segment of the chromosome

Fusions - when two genes come together to form one new hybrid gene

Duplications - copy of a region of the chromosome

Mutagens

physical or chemical agents that can cause mutations

• radiation, x-rays, UV

• Carcinogens

Spontaneous mutations

can occur during DNA replication, recombination, or repair