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Transcription Fig.12.4
Only a small region of DNA is transcribed, typically between 100 and 10000 nucleotides long. The enzyme RNA polymerase catalyzes the formation of the phosphodiester bond between the nucleotides, leading to the assembly of RNA. Transcription starts with RNA polymerase binding to a specific nucleotide sequence on the DNA, called promoter. RNA polymerase alone does not recognize the site, several so called initiation factors (proteins) are necessary participants. Only after the enzyme is tightly bound to the DNA the RNA synthesis begins with the addition of ribonucleotides. They need to be in their activated form as triphosphates (UTP, CTP, ATP, GTP). As seen above, the first DNA nucleotide to be transcribed is thymidine (T). The complementary nucleotide which is inserted into the growing RNA chain is adenosine, because no other base (U,C or G) can form H-bonds with T. The next base in the template DNA is A. If a complementary DNA strand would be assembled, the base used would be T. Since RNA has no thymidine nucleotide (T), uracil (U) is used. Uracil will pair with adenine (A) in a similar fashion as T. Thus the second nucleotide in the growing m-RNA chain is uridine (U). The next nucleotide is G and so on. Approximately 40 nucleotides are added per second. Synthesis is terminated when a DNA sequence is encountered that destabilizes the RNA polymerase complex. After the synthesis is completed m-RNA undergoes a number of modification: addition of a 5’ cap (facilitates the initiation phase in protein synthesis), addition of a poly A tail (30 to 200 adenine nucleotides) and the excision of certain RNA segments from the m-RNA.
In prokaroytic organisms the m-RNA is an exact complementary copy of the
gene inscribed in DNA. In eukaryotic organisms certain sections of the
newly synthesized m-RNA (called introns) are excised by specific enzymes (intron/exon ).
The remaining sections are called exons and they make up the final m-RNA,
whose sequence is translated into protein. Introns are formed because
certain sections of the m-RNA are self complementary and they can form
hair pins. (these hair pins are not recognized as a termination signals by
the RNA polymerase). Depending on what enzymes are present some hairpins
are not excised. Thus, the same structural gene can produce different
m-RNAs (= different proteins) depending on what excision enzymes are
active.
After these modifications, the m-RNA leaves the nucleus and diffuses into the cytoplasm.
Transfer RNA
In addition to the m-RNA which carries in its base sequence the information on the amino acid composition of the protein to be made, a second type of RNA participates in protein synthesis, called transfer RNA (t-RNA) . Each protein amino acid has its own specific t-RNA. They function as is transport vehicle for the amino acids and align themselves along the m-RNA at positions defined by the base sequence of the m-RNA. In a mechanism similar to m-RNA, t-RNA is synthesized in the nucleus by transcription of a specific DNA segment.
While m-RNA consists of a long strand of nucleotides, t-RNAs are small molecules, containing only 75 to 95 nucleotides, which are folded into a "cloverleaf" secondary structure as shown to the left. It contains four double helical structural elements. In addition to the four RNA bases (A, U, G and C) t-RNA contains unusual bases such as Inosine (I), Methylinosine (ml) , Pseudouridine (psi), Dihydrouridine (D) , Ribothymidine (T) and Methylguanosine (Gm). The t-RNA for alanine is shown above.
The region called anticodon is a trinucleodite sequence located on one of the three loops. The attachment of e.g. Ala to its specific t-RNA
( the one which has the anticodon C G I ) is carried out in the cytoplasm by an enzyme which recognizes both, the specific t-RNA and Ala. The amino acid is attached to the 3’ end of the t-RNA. The t-RNA which carries the amino acid Phe has an anticodon of A A G and Trp (tryptophan) is carried by its specific t-RNA, which has an anticodon of A C C. The anticodon of the t-RNAs is complementary to a trinucleotide sequence on a m-RNA. This is shown in the drawing below.
The trinucleotide sequence G C C on the m-RNA can bind only to the complementary anticodon bases of the Ala-t-RNA, that means, among all amino acyl t-RNAs only Ala-t-RNA can be positioned at this location i.e. only a Ala-t-RNA will recognize the sequence G C C. The next trinucleotide on the m-RNA is U U C and only a t-RNA carrying the amino acid Phe with the anticodon A A G can bind to this location. As you already can see the sequence of nucleotides on the m-RNA determines the positioning of the amino acyle t-RNAs and consequently amino acids (in our case Ala, Phe, Trp).
Genetic Code
Fig. 12.5 Genetic Code The trinucleotides on the m-RNA are called codon. For example the second trinucleotide UUC on the m-RNA represents a code for Phe since only the Phe-t-RNA with an anticodon A A G can associate with it. But Phe-t-RNA will also bind to a m-RNA trinucleotide UUU . This somewhat unspecific binding between the third base of the t-RNA (called wobble base, as seen in the above figure of
Ala-t-RNA ) and the corresponding nucleotide on the m-RNA is caused by the extended geometry of the anticodon region. While the first two bases follow the complementary binding rules, the third base can pair with a number of different bases: For example, the wobble base (third base of the anticodon) in Phe-t-RNA is G. In this position G can bind to either C or U. Thus the codon (trinucleotide sequence on m-RMA) for Phe can either be UUC or UUU. The t-RNA for Ala has an anticodon CGI with inosine ( I ) as wobble base. Inosine in this position can pair with U, C or A and to some extend with G. Thus, Ala has four possible codons : GCU, GCC, GCA and GCG. With a few exceptions each of the 21 amino acids have 2 or 4 codons. Because of such multiple assignments the code is said to be degenerate. Since there are 3 bases per codon, the total number of possible base combinations is 64. All but 3 codons are used for amino acids. These three codons - UAA, UAG and UGA - are termination codons. As we shall see later, when they are encountered in protein synthesis, the synthesis stops. The genetic code is universal, which suggest that the earliest life forms had already developed the code.
Amino acyl t-RNA and m-RNA on their own can not synthesize protein, they need the ribosomes. Ribosomes are either freely suspended in the cytoplasm , but they also can bind to the membrane of the endoplasmic reticulum as we shall see later. Ribosomes are made up of ribosomal RNA (r-RNA) and protein and consist of a small subunit and a large subunit, which can dissociate or associate depending on the Mg++ concentration.
A simplified model of protein synthesis (also called translation ) is shown below
Initiation Fig.12.10 Elongation Fig.12.11 Elongation Fig.12.11cont. Termination Fig.12.12
In the initiation stage of transcription t-RNA binds to the small ribosomal subunit. Next the m-RNA binds to t-RNA/small subunit complex in such a way that the anticondon of the t-RNA is aligned with the starting trinucleotide sequence (codon) on the m-RNA. In most eukaryotic m-RNAs protein synthesis starts with the AUG codon, which codes for the amino acid Met. Thus, proteins have Met as N-terminal amino acid. However, after the synthesis is completed, Met is removed again.
After t-RNA, m-RNA and the small ribosomal subunit are combined, the large subunit binds to the complex together with enzymes called initiation factors. The polypeptide synthesis begins with the assistance of certain elongation factors. Once two amino acyl t-RNAs are positioned next to each other in the P-site and A-site of the ribosome a peptide bond is formed between them. The ribosome complex moves forward on the m-RNA to the next codon, connecting the next amino acid to the growing chain. The process is repeated until a stop codon is encountered, which causes the ribosomal complex to dislodge from the m-RNA and thus the synthesis is terminated.
Mutations
Silent (synonymous) Mutation (add. image)
Point (missense) Mutation (add. image)
Point (nonsense) Mutaion
Frame Shift Mutation
Sickle Cell
