Molecular Genetics XV

 

Transcription

 

Prokaryotes

At signal sequences called promoters, the RNA polymerase will separate the strands and use one strand as a template.  A portion of the enzyme (sigma factor) dissociates when the RNAse binds to the promoter. Transcription can then begin. The RNAse will link incoming NTPs according to complementarity rules. A mRNA in a prokaryote may contain one or several genes to be translated immediately into polypeptides.

 

Eukaryotes

In eukaryotes, different RNA polymerases are used to transcribe the different types of RNA. We’ll just look at the transcription of mRNA by its associated enzyme RNA polymerase II.

 

Initiation

 

The RNAse II binds to the DNA at a promoter region, but will not begin transcription unless a specific molecule is bound to a sequence called a TATA box.  These molecules are called transcription factors. The two DNA strands separate and transcription begins.

 

Elongation

 

RNAse II is self-priming.  As it moves along the coding strand, it incorporates incoming NTPs and also opens up the double helix. Synthesis must occur in a 5’ to 3’ direction. The growing strand is anti-parallel to the template. So the DNA strand is read in a 3’ -5’ direction. The rate of elongation is 30-60 NTPs per second. As the pre-mRNA molecule grows, it peels away from the DNA strand and the double helix reforms behind the moving RNAse II. Sometimes a series of RNAse II molecules will transcribe the same gene, one after another.

 

Termination

 

At a signal sequence on the coding strand, the RNAse II will disengage from the DNA. Transcription stops.  Translation cannot begin until the pre-mRNA is processed and exits the nucleus as a mature mRNA.

 

 

RNA processing

 

Two important modifications must be made to the pre-mRNA transcript before it exits the nucleus.

 

Splicing. Intervening sequences (introns) that do not code must be spliced out from the transcript. This is accomplished by a spliceosome (enzymes, snRNAs, and snRPs). The expressing sequences (exons) are spliced together.

Faulty splicing has been linked to such diseases as  β-thalassemia and systemic  lupus erythematosis.

(Take home: introns go out, exons stay in. Go figure!)

 

5’ cap and poly-A tail. A modified GTP cap is placed backwards onto the 5’ end of the transcript to protect it from degradation. This may also help with recognition by the ribosomal subunits. The 3’ end of the transcript is modified by the addition of 150-200 adenine nucleotides. This helps to export the mRNA from the nucleus and also helps to protect the message from hydrolytic enzymes.

 

Activating tRNA

 

To build a polypeptide, amino acids must be brought in and placed in the proper order.  Transfer RNA molecules are adaptor molecules, linking the mRNA codon to its proper amino acid.  After transcription, the tRNA molecules travel to the cytoplasm where translation will occur.  The cloverleaf shape of the tRNA reveals important structures.

The 3’ end that overhangs the 5’ end has a CCA sequence that is used for amino acid attachment.

At the bottom loop of the tRNA lie three nucleotides that make up the anticodon. The anticodon is the complement of the mRNA codon.

There are only about 45 tRNA molecules even though there are 64 codons.

Why?

3 codons don’t code for anything (UGA, UAA, and UAG)

There is an exception to base pairing rules called wobble.

 

Wobble describes the ability of a tRNA to recognize two or three codons that specify the same amino acid. This “wobble” occurs at the third base of the mRNA codon (Can you see why this makes more sense than the first or second base?).  The base U in the wobble position can pair with either A or G on the mRNA. Some tRNAs have a modified base, inosine (I), that can pair with either U, C, or A in the wobble position.

Example: a tRNA with the anticodon CCI can pair with GGU, GGC, or GGA that all code for the amino acid glycine. “Cells are thrifty”

Activation involves an enzyme (aminoacyl-tRNA transferase) that is specific to an amino acid. The amino acid with ATP recognizes the correct enzyme. Two phosphate groups come off (energy source) and the amino acid binds to the AMP.  The tRNA with the correct anticodon for that amino acid will dock onto the enzyme and displace the AMP. The tRNA with its amino acid will leave the enzyme and head for the ribosomes.

 

Ribosomes

 

The structure of ribosomes is also vital to their function.  Ribosomes act as a workbench for polypeptide synthesis but also as a “keeper of the reading frame”.

Ribosomes are made of 60% RNA and 40% protein. A small and large subunit are assembled in the nucleolus and exported to the cytoplasm. The two subunits come together only when translation occurs. The large subunit has two tRNA binding sites. The P site holds the tRNA holding the growing polypeptide. The A site holds the incoming tRNA with its single amino acid. New research suggests that there is an E site for exit. The small subunit has a mRNA binding site.

 

Translation

 

Translation occurs in the cytoplasm and involves ribosomes, mRNA, tRNA, enzymes, factors, and energy. We can look at translation in three steps: initiation, elongation, and termination.

 

Initiation

 

The small ribosomal subunit will bind to an initiator tRNA carrying methionine (the first amino acid of the polypeptide). The mRNA binds to a recognition site on the small subunit and its initiation codon, AUG, will base pair with the UAC on the met-tRNA. Initiation factors also participate and GTP acts as an energy source to attach the large ribosomal subunit. When the large subunit engages, the met-tRNA fits inside the P site.