D1.2

Transcription

Three main steps: initiation, elongation and termination

• Initiation: RNA polymerase binds to the promoter. DNA unwinds and separates into strands

• Elongation: RNA polymerase moves along the coding sequence, synthesising RNA in a 5’ → 3’ direction

• Termination: RNA polymerase reaches the terminator, both the enzyme and mRNA strand detach and the DNA rewinds

• Free nucleotides exist in the cell as nucleoside triphosphates (NTPs), which line up opposite their complementary base partner

• RNA polymerase covalently binds the NTPs together in a reaction that involves the release of the two additional phosphates

• The 5’-phosphate is linked to the 3’-end of the growing mRNA strand, hence transcription occurs in a 5’ → 3’ direction

DNA stability

DNA is a complex molecule used endlessly in gene expression. It is repeatedly and continually being broken down by RNA polymerase for transcription and then rewinding again and again. 

DNA in eukaryotes is not “naked”. It is supported by proteins called histones.  

In addition to this there are enzymes called gyrase that help to support the DNA when it is unwound in transcription. 

This is particularly important in cells like neurons that never undergo mitosis. As such the DNA is never recreated new as it would be for any other cells. 

So neuronal DNA must remain intact for the life of the individual. 

Histones

Histones are proteins that form structural units known as nucleosomes that help to support the structure of DNA for the life of the organism.  

Genes that are rarely/never expressed are bound tightly to the histone proteins. 

Cells can increase the rate of transcription by altering the binding of DNA / Histones and making it easier/faster for RNA polymerase to work its way along the DNA Strand. 

Gene Expression

Coding DNA Mutations have almost no impact on protein synthesis and the shape of the protein. 

Non-coding DNA mutations can completely stop transcription as RNA polymerase binding is affected. 

By blocking the start codons RNA Polymerase can not bind and genes can be switched off and so never transcribed.

No Transcription = No Translation = No protein!

The enzymes that run transcription have a binding site known as a “promoter region”  at the start of the gene. 

If your cells block this region, transcription will be prevented. 

Translation

mRNA binds to ribosome.

The ribosome is comprised of two separate parts. 

The mRNA binds to the small ribosomal subunit. 

The large subunit then binds simultaneously. The large unit contains active sites for the formation of the new protein molecule. 

tRNA brings in amino acids. 

Each Amino acid is represented by a triplet (3BP = 1AA) 

tRNA has the matching anticodon. 

A polypeptide chain if formed. 

Universality of DNA

n modern farming and medicine we use genetic modification increasingly often. This is only possible because of the universality of the genetic code. 

All organisms use the same bases and most organisms on Earth use the same set of degenerate codes to represent the same amino acids. 

This means that we can take genes from a Human and place them in bacteria or sunflowers to grow human proteins such as hormones or enzymes. 

We can also take genes from fungi and place them in plants for antibiotic resistance. 

Cause of sickle cell anaemia

Haemoglobin is formed from 4 polypeptide chains (two alpha and two beta). The beta protein is very slightly different…

The sixth DNA triplet is altered from CTC 🡪 CAC. This means that glutamic acid is switched for valine.

Non-coding DNA

Need to know 4 examples:

• Gene Promoter regions - the binding site for RNA polymerase.

  
  

  Telomeres - as DNA is replicated and primers are removed a tiny section at the end of each chromosome is lost. Telomeres cap the ends of chromosomes and allow for this degradation by extending the DNA molecule beyond the genes.

  
  

  Genes for tRNA and rRNA - the RNA formed is never expressed in to a protein and so is considered non-coding. 

  
  

  Introns - Sections between Exons - Exons code for subsections of protein strands in a gene 

  
  

  Exons = expressed - produce a protein Introns = not expressed

  
  

Post transcriptional Modification

Newly produced mRNA needs to be changed into mature mRNA. Post-transcriptional modification happens before the mRNA leaves the nucleus. 

Firstly a 5’ cap is added. This is a modified Guanine molecule. It stabilises the mRNA and helps it to bind to the ribosome. 

At the other end of the mRNA a poly A tail is added to the 3’ end. Translation stops before the poly A tail. 

The poly A tail helps to stabilise the mRNA and is used in transporting the molecule to the ribosome. 

mRNA is now mature…

Alternate Gene Splicing

The same pre-mRNA can be spliced in many different ways.

Some exons may:

• be included in the final mRNA

• be skipped and removes

As a result, different mature mRNA’s can be produced from the same gene.

Why is this important?

This is important because:

• increases the number of proteins and organism can produce

• allows a single gene to have multiple function

• contributes to the complexity of eukaryotes

• different tissues can produce different protein variants from the same gene

“One gene can produce more than one protein because exons can be combined in different ways during RNA processing”

Initiation of Translation

1. Initiation is the first stage of translation. 

2. An activating enzyme attaches to the amino acid methionine to an initiator tRNA (anticodon UAC).

3. Initiator tRNA attaches to the small subunit of the ribosome in the p site. This produces a ternary complex (made of three parts).

4. Ternary complex binds to the 5’ end of the mRNA and using the anticodon scans along to the start codon AUG. 

5. The anticodon and codon are linked by hydrogen bonds. 

6. The large ribosomal subunit binds. 

7. A tRNA with an anticodon complementary to the next codon on the mRNA binds at the A site. 

8. Peptide bond forms. The tRNA in the A site holds a dipeptide. The ribosome moves in a 5’-->3’ direction by three bases. Initiator tRNA moves to the E site and exits. 

Elongation of the polypeptide

The covalent (peptide) bond is formed between the carboxyl group in the P site and the amino acid  in the A sites. 

The ribosome then moves along the mRNA strand 5’ to 3’. 

This means the methionine in the P site is now in the exit site (E) and so breaks off and leaves. 

The amino acid formerly in the A site is now in the P site and as another Amino acid joins the A site a new bond is formed. 

As the mRNA moves through the ribosome this is known as transcriptional translocation. So the protein strand gets longer. This is known as elongation.

Termination of Translation

There are 3 stop codons UAA, UAG and UGA. 

There is no corresponding amino acid to these codes and so translation will cease. 

When the ribosome reaches the stop codon the polypeptide is released.

Polypeptide Modification

Named example of SPEC - Insulin

Occurs in the Golgi apparatus, but some modification can occur in the RER

Preproinsulin turns to proinsulin (RER)

Proinsulin turns to insulin (GOLGI)

• Preproinsulin is produced on a ribosome attached to RER. 

• 110 amino acids long. 

• Goes into the lumen of the RER.

• 24 amino acids removed from the N-terminal end leaving 86 amino acids called Proinsulin. 

• Proinsulin folds with 3 x disulphide bonds stabilizing the tertiary structure. 

• Some peptide bonds are broken allowing 33 amino acids to be removed leaving:

  • A-chain with 21 amino acids. 

  • B-chain with 32 amino acids. 

• A pair of amino acids are removed from the C terminal end of the B-chain making mature insulin with a total of 51 amino acids. 

Protein Recycling

The total protein content of the cell is known as the proteome. 

Protein synthesis has a very high energy cost and amino acids can not be stored unless they are in a protein. 

So what happens to all the unwanted polypeptide chains? 

All unwanted polypeptides including denatured enzymes etc. are continually broken down and the amino acids recycled to make new proteins. 

The enzymes which carry out this recycling process are known as proteasomes. 

Proteomes = all proteins in a cell (enzymes, structural, hormones, membrane proteins etc. etc.) 

Proteasome = enzymes that recycle proteins