Molecular Biology II

Transcription and Translation (aka Gene Expression)

Learning Objectives

Gene Expression

• The combined process of transcription of a gene into mRNA, the processing of that mRNA, and its translation into protein

• Required for:

  • Adaptation

  • Tissue specific differentiation

  • Development

RNA Transcription

• Transcribe DNA into RNA (slightly different language)

• dsDNA → ssRNA

RNA polymerase

• enzyme that catalyzes the reaction of rNTP polymerization

• always synthesized in the 5’ → 3’ direction

RNA polymerase in prokaryotes

•The sigma region of the bacterial RNA polymerase recognizes specific regions of DNA called promoters

•At approximately 10 and 35 base pairs from the start site there are similar DNA sequences termed the -10 and -35 sequence

Which region of bacterial RNA polymerase recognizes specific regions of DNA called promoters?

• the sigma region

  • At approximately 10 and 35 base pairs from the start site there are similar DNA sequences termed the -10 and -35 sequence

In other words, …

• Bacterial RNA polymerase recognizes specific DNA sequences known as promoters, particularly the -10 (Pribnow box) and -35 consensus sequences, which are upstream of the transcription start site.

• The recognition is primarily mediated by the sigma (σ) subunit of the RNA polymerase, which binds to these sequences and facilitates the opening of the DNA double helix to begin transcription

RNA polymerase in Eukaryotes

• The majority of eukaryotes initiate transcription using the TATA box region at approximately -30bp

• TATA box consensus sequence is recognized by the RNA polymerase and directs it where to initiate transcription

  • Pol I  : large ribosomal RNA

  • Pol II: messenger RNA (mRNA)

  • Pol III: small rRNA, tRNA and other small RNA

TATA box

• a DNA sequence, most commonly TATAAA, located in the promoter region of many genes, about 25-35 base pairs upstream (-30bp) of the transcription start site.

• It serves as a recognition site for the TATA-binding protein (TBP), a key transcription factor that recruits RNA polymerase and initiates transcription.

• The TATA box's sequence is rich in adenine and thymine (A-T), which allows for easier unwinding of the DNA strands for transcription.  

Coding region in Eukaryotes

• region that contains the DNA sequence that is transcribed into mRNA, which is ultimately translated into protein

Regulatory region in Eukaryotes

• region that consists of two classes of elements

  • one class is responsible for ensuring basal expression

    → these elements generally have two components (the proximal component = TATA box or Inr or DBP elements direct RNA polymerase to the correct site. In TATA-less promoters, an initiator (Inr) element that spans the initiation site may direct the polymerase to the appropriate site. The upstream elements, specifies the frequency of initiations, for example the CAAT box.

  • the other class is responsible for ensuring regulated expression

    → the distal regulatory elements consist of enhancers and repressors and other regulatory regions. Enhancers and repressors regulate transcription expression based on various signals including hormones, heat shock, heavy metals, and chemicals

The proximal promoter elements are required for:

a. basal expression of a gene

b. up regulation of a gene

c. down regulation of a gene

d. allowing transcription factors and RNA polymerase access to the DNA template

e. none of the above

a. basal expression of a gene

• basal expression refers to the baseline level of gene transcription that occurs even without strong activating signals.

• It is established by the core promoter and the binding of general transcription factors (GTFs) to recruit RNA polymerase to initiate transcription at a low but consistent level. This basal rate serves as a foundation upon which other regulatory mechanisms, such as enhancers and enhancers and repressors, can build to increase or decrease gene activity as needed

Pol I

• one of the 3 RNA polymerases in eukaryotes

• large ribosomal RNA

Pol II

• one of the 3 RNA polymerases in eukaryotes

• messenger RNA (mRNA)

• often uses the TATA box consensus sequence to initiate transcription

Pol III

• one of the 3 RNA polymerases in eukaryotes

• small rRNA, tRNA, and other small RNA

RNA polymerase

• the primary enzyme responsible for transcription (always in the 5^’-3^’ direction)

Proximal promoter elements

Basal expression in eukaryotes is controlled by _____ ______ _____

Distal regulatory elements

Regulated expression in eukaryotes is controlled by ____ _____ ______

What are the three steps of transcription?

1. Initiation = promoter, transcription factors and start point

2. Elongation = DNA is unwound, the template strand is read in the 3^’-5^’ direction and RNA is synthesized in the 5^’-3^’ direction by RNA polymerase

3. Termination = stop signal causes RNA polymerase to dissociate and the RNA transcript is complete

Initiation

•RNA polymerase recognizes and binds a specific promoter site

•RNA polymerase separates the DNA strands to expose the template and create the transcription bubble

•Initiation is complete when the first two ribonucleotides of an RNA chain are linked by a phosphodiester bond

Elongation

• occurs along the DNA molecule and therefore DNA unwinding must occur

• Transcription bubble is approximately 20 base pairs per polymerase molecule

• RNA polymerase has “unwindase” activity that opens the DNA helix

• Topoisomerases precede and follow the progressing polymerase to prevent super helical complexes

• RNA is elongated in the 5’-3’ direction, therefore DNA is read in the 3’-5’ direction

Termination

• Certain factors cause the RNA polymerase to dissociate

  • Rho dependent, rho independent (prokaryotes)

  • poly A signal (eukaryotes)

• Pre mRNA is released (but must still be processed)

Rho-Dependent termination

• occurs in prokaryotes

• a sequence in the DNA molecule is recognized by a termination protein (the rho factor)

  • Rho is an ATP-dependent RNA stimulated helicase that disrupts the nascent RNA-DNA complex and causes RNApolymerase to dissociate from the template DNA.

Rho-Independent Termination

• occurs in prokaryotes

• process that requires the presence of intrachain self-complementary sequences in the newly formed primary transcript so that it can acquire a stable hair pin turn that slows down the progress of the RNA polymerase and causes it to pause temporarily

  • near the stem of the hairpin, a sequence occurs that is rich in G and C (this stabilizes the secondary structure of the hair pin)

  • following the hair pin is a series of Us which provides a section of weak U-A bonding that facilitates the dissociation of the primary transcript from the DNA

Downstream Terminator sequence

• occurs in eukaryotes

• poly-A sequence

• termination in eukaryotes is less well understood

Post-transcriptional modification

In eukaryotes, mRNA must be modified before it is ready for transport to the cytoplasm for translation into proteins

1. The 5’ end of the RNA becomes capped (a methylated guanine residue is added to the end of the RNA transcript)

2. The 3’ end is modified by the addition of about 200 adenines by the poly-A polymerase

3. The RNA must undergo splicing to remove the introns

RNA splicing

• one of the three post-transcriptional modifications that occur in eukaryotic cells

  • RNA is spliced to remove introns

  • alternative splicing can provide different protein products as well as a mechanism for differential regulation of the same protein

Methylated guanine cap

• one of the three post-transcriptional modifications that occur in eukaryotic cells

  • it is added to the 5^’ end of the mRNA

  • ribosomal subunits recognize the 5^’ cap and use it to initiate translation

Poly A tail

• one of the three post-transcriptional modifications that occur in eukaryotic cells

  • we take advantage of this in the lab and can use an oligo dT primer when examining expression levels of mRNA for use in PCR

How does histone modification increase or decrease transcription?

• Two post-translational modifications

  1. acetylation = increases access to DNA, increases transcription

  2. histone or DNA methylation = decreases access to DNA, decreases transcription

Acetylation

• a type of histone modification

• increases access to DNA, increases transcription

  1. Histone Acetyl Transferase (HAT)

  2. Histone Deacetylase (HDAC)

Histone or DNA methylation

• a type of histone modification

• decreases access to DNA, decreases transcription

  1. DNA methyl transferase (DNMT)

  2. Demethylase

Start codon

AUG = Met

What are the three key components of translation?

• tRNA

• ribosomes

• mRNA

tRNA

• responsible for transferring the amino acids to the ribosome

• there are two important sites to remember on the molecule, the amino acid attachment (amino acyl) site and the anti-codon site

• tRNA carries the amino acid and recognizes the codon

Ribosome

• Site of protein synthesis

• Has two binding sites for tRNA molecule: A and P sites, each of which extends over both subunits.

• During translation, the A site binds an incoming Aminoacyl tRNA as directed by the codon currently occupying the site. The codon specifies the next amino acid to be added to the growing peptide chain.

• The P site codon is occupied by the Peptidyl-tRNA. This tRNA carries the chain of amino acids that has already been synthesized.

• An E site is also there that is occupied by the empty tRNA that is about to exit the ribosome

What are the steps of translation?

Requires two ribosomal subunits, mRNA, tRNA, and (in prokaryotes) 3 initiation factors.

• First, the small ribosomal subunit binds to an initiation factor which then binds to the mRNA at the Shine-Dalgarno sequence (a specific sequence that the ribosomal subunit recognizes in prokaryotes).

• Next IF-2 binds to MET-tRNA and promotes binding to the start codon.

• The ribosome scans the mRNA until it encounters the start codon. This is called the initiation complex.

• Next the large subunit attaches, the initiation factors are released and the complex is ready for protein synthesis.

• Peptidyl transferase catalyzes the formation of a peptide bond.

• The whole ribosome shifts over one codon and the A site is now free to accept the tRNA.

The peptide chain elongation process terminates when the stop codon is encountered on the mRNA. In prokaryotes, 15 aa can be added/sec.

Anti-codon site

• a part of tRNA

• 3 bases that bind to the RNA and pair with the codon of the mRNA. Indicates which amino acid will be added to the peptide chain

Initiation phase of translation

• mRNA comes from transcription which is used to build a protein in the process of translation

  • A ribosome and initiator tRNA bind to the start codon on the mRNA

1. First, the small ribosomal subunit binds to an initiation factor which then binds to the mRNA at the Shine-Dalgarno sequence (a specific sequence that the ribosomal subunit recognizes in prokaryotes.)

2. First, the small ribosomal subunit binds to an initiation factor which then binds to the mRNA at the Shine-Dalgarno sequence (a specific sequence that the ribosomal subunit recognizes in prokaryotes.

   Next the large subunit attaches, the initiation factors are released and the complex is ready for protein synthesis.

3. Next the large subunit attaches, the initiation factors are released and the complex is ready for protein synthesis.

Elongation / Termination phase of translation

• During translation, the A site binds an incoming Aminoacyl tRNA as directed by the codon currently occupying the site. The codon specifies the next amino acid to be added to the growing peptide chain.

• The P site codon is occupied by the Peptidyl-tRNA. This tRNA carries the chain of amino acids that has already been synthesized.

• An E site is also there that is occupied by the empty tRNA that is about to exit the ribosome

****The peptide chain elongation process terminates when the stop codon is encountered on the mRNA. In prokaryotes, 15 aa can be added/sec.

Transcription vs Translation Summary

• Transcription involves initiation, where RNA polymerase binds to DNA to start copying a gene's code into messenger RNA (mRNA), followed by elongation, where RNA polymerase synthesizes the mRNA strand, and termination, where it stops and the mRNA is released.

• Translation then uses this mRNA to build a protein, also in three phases: initiation, with a ribosome and initiator tRNA binding to the start codon on the mRNA; elongation, where amino acids are linked to form a growing polypeptide chain; and termination, where a stop codon signals the release of the finished protein

Prokaryotic vs Eukaryotic Translation

• Prokaryotes = ribosomes recognize a sequence called the Shine-Dalgarno sequence, where they bind to initiate translation

• Eukaryotes = ribosomal subunits  recognize and bind to the 5’ cap

• In eukaryotes there are many more protein factors involved (there are 3 Ifs in prokaryotes and at least 10 in eukaryotes)

• In eukaryotes, the poly A tail likely increases rate of re-initiation

mRNA

• the code that dictates the order of amino acids in a protein

Amino acyl site

• a part of the tRNA

• carries a specific amino acid based on the anti-codon site. A tRNA molecule can only care one specific amino acid