TRANSCRIPTION (DNA  RNA)

Transfer of information from DNA molecule to RNA molecule. DNA  RNA Transcription occur in the nucleus for eukaryotes and in the cytoplasm for prokaryotes. Primary product of transcription is three major types of RNA: mRNA, tRNA, rRNA. Transcription

Transcription process consist of 3 stages: INITIATION ELONGATION TERMINATION

INITIATION OF TRANSCRIPTION Transcription will start at the promoter region located at the beginning of the gene. the enzyme that synthesize RNA is RNA polymerase. to initiate transcription, RNA polymerase must first locate the beginning of the gene.

near at the beginning of every gene, there is a region known as the promoter. Transcription will start at this promoter region. Promoter is an untranscribed sequence of DNA bases. It usually consist of one or more repetitions of the sequence TATA. when RNA polymerase binds to the promoter region, the DNA double helix at the beginning of the gene unwinds and transcription begins.

ELONGATION OF TRANSCRIPTION PROCEEDS UNTIL RNA POLYMERASE REACHES A TERMINATION SIGNAL the ‘body’ of the gene is where elongation of the RNA strand occurs. RNA polymerase then travels down one of the DNA strands, called the template strand, synthesizing a single strand of RNA with bases complementary to those in the DNA. base pairing between RNA and DNA is the same as between two strands of DNA, except that uracil in RNA pairs with adenine in DNA.

after about 10 nucleotides have been added to the growing RNA chain, the first nucleotides in the RNA molecule separate from the DNA template strand. this separation allows the two dDNA to rewind into a double helix thus, as transcription continues to elongate the RNA molecule, one end of the RNA drifts away from the DNA; RNA polymerase keeps the other end temporarily attached to the DNA template strand.

RNA polymerase continues along the template strand of the gene until it reaches a sequence of the DNA bases known as termination signal. at this point RNA polymerase release the completed RNA molecules and detaches from the DNA.

RNA molecule almost always consists of a single strand. In DNA or RNA, the four nucleotide monomers act like the letters of the alphabet to communicate information. To get from DNA, written in one chemical language, to protein, written in another, requires two major stages, transcription and translation. blocks of three nucleotides (codons), are decoded into a sequence of amino acids. It would take at least 300 nucleotides to code for a polypeptide that is 100 amino acids long.

How is RNA modified after transcription? In prokaryotes, the RNA copy of a gene (mRNA) is ready to be translated into protein. In fact, translation starts even before transcription is finished. In eukaryotes, the primary RNA transcript of a gene needs further processing before it can be translated. This step is called “RNA processing”. Also, it needs to be transported out of the nucleus into the cytoplasm. Steps in RNA processing: 1. Add a cap to the 5’ end 2. Add a poly-A tail to the 3’ end 3. splice out introns. How is RNA modified after transcription?

Capping RNA produced from transcription is unstable, especially at the ends. The ends are modified to protect it. At the 5’ end, a slightly modified guanine (7-methyl G) is attached “backwards”, by a 5’ to 5’ linkage, to the triphosphates of the first transcribed base. At the 3’ end, the primary transcript RNA is cut at a specific site and 100-200 adenine nucleotides are attached: the poly-A tail. These A’s are not coded in the DNA of the gene.

Introns are regions within a gene that don’t code for protein and don’t appear in the final mRNA molecule. Protein-coding sections of a gene (called exons) are interrupted by introns. The function of introns remains unclear. There are a few prokaryotic examples, but most introns are found in eukaryotes. Introns

Intron Splicing Introns are removed from the primary RNA transcript while it is still in the nucleus. Introns are “spliced out” by RNA/protein hybrids called “spliceosomes”. The intron sequences are removed, and the remaining ends are re-attached so the final RNA consists of exons only.

Summary of RNA processing In eukaryotes, RNA polymerase produces a “primary transcript”, an exact RNA copy of the gene. A cap is put on the 5’ end. The RNA is terminated and poly-A is added to the 3’ end. All introns are spliced out. At this point, the RNA can be called messenger RNA. It is then transported out of the nucleus into the cytoplasm, where it is translated.

HOW IS THE BASE SEQUENCE OF A MESSENGER RNA MOLECULE TRANSLATED INTO PROTEIN??

MESSENGER RNA (mRNA) CARRIES THE CODE FOR PROTEIN SYNTHESIS FROM NUCLEUS TO THE CYTOPLASMA All RNA is produced by transcription of DNA, but only mRNA carries the code for the amino acid sequence of a protein. In the cytoplasm, mRNA binds to ribosomes, which synthesize a protein specified by the mRNA base sequence.

Ribosomes consist of two subunits, each composed of Ribosomal RNA (rRNA) and Protein Ribosomes, the structures that carry out translation, are composed of rRNA and many different proteins. Each ribosome is composed of two subunits: small & large. The small subunit has binding sites for mRNA, a ‘start’(methionine) tRNA, and several others proteins that collectively make up the ‘initiation complex’. The large subunit has binding sites for two tRNA molecules and catalytic site for joining together the amino acids attached to the tRNA molecules. During protein synthesis, the small and large subunits come together and sandwich an mRNA molecules between them.

Transfer RNA (tRNA) Molecules Decode the Sequence of Bases in mRNA into the Amino Acid Sequence of a protein The ability of tRNA to deliver the proper amino acid depends on specific base pairing between tRNA and mRNA. Each tRNA has three exposed bases, called – ANTICODON, which form base pairs with the mRNA codon. For example, the mRNA codon AUG forms base pairs with the anticodon UAC of a tRNA --- that has the amino acid methionine attached to its end ---incorporate into growing protein. If the codon on mRNA is UUU, a tRNA with an AAA anticodon and carrying phenyalanine will bind to it.

Transfer RNA tRNA molecules are short RNAs that fold into a cloverleaf pattern. Each tRNA has 3 bases that make up the anticodon. These bases pair with the 3 bases of the codon on mRNA during translation. Each tRNA has its matching amino acid attached to the 3’ end. A set of enzymes, the “aminoacyl tRNA synthetases”, are used to “charge” the tRNA with the proper amino acid.

HOW DOES THE CELL RECOGNIZE WHERE CODONS START AND STOP? All protein originally begin with the same amino acid, methionine – specified by the codon –AUG (aka start codon). Three stop codons: UAG, UAA and UGA will signal the termination of translation. When the ribosome encounters a stop codon, it release both the newly synthesized protein and the mRNA. This establishes the reading frame and subsequent codons are read in groups of three nucleotides. In summary, genetic information is encoded as a sequence of codons, each of which is translated into a specific amino acid during protein synthesis

The Genetic Code Each group of 3 nucleotides on the mRNA is a codon. Since there are 4 bases, there are 43 = 64 possible codons, which must code for 20 different amino acids. More than one codon is used for most amino acids (Several codons can specify the same amino acid): the genetic code is “degenerate”. This means that it is not possible to take a protein sequence and deduce exactly the base sequence of the gene it came from. AUG is used as the start codon. All proteins are initially translated with methionine in the first position, although it is often removed after translation. There are also internal methionines in most proteins, coded by the same AUG codon. There are 3 stop codons, also called “nonsense” codons. Proteins end in a stop codon, which codes for no amino acid (UAA, UAG, UGA).

The genetic code is almost universal The genetic code is almost universal. It is used in both prokaryotes and eukaryotes. However, some variants exist, mostly in mitochondria which have very few genes. For instance, CUA codes for leucine in the universal code, but in yeast mitochondria it codes for threonine. Similarly, AGA codes for arginine in the universal code, but in human and Drosophila mitochondria it is a stop codon.

TRANSLATION PROCESS Three stages of translation : 1) Initiation of protein synthesis 2) Elongation of the protein chain 3) Termination

Initiation of Translation The first AUG codon (codes for methionine) in an mRNA sequence specifies the start of translation. An initiation complex consist of small ribosomal subunit, a methionine tRNA and several others protein. This initiation complex will bind to a molecule of mRNA with the AUG start codon.

Positioning of mRNA is signaled by a ribosome recognition sequence on the mRNA. the initiator tRNA (carrying the methionine amino acid) with the UAC anticodon will bind to the AUG codon at the mRNA.

Large ribosomal subunit then joins the initiation complex. Initiator tRNA is in the P site of large ribosomal subunit. The A site is available for the next tRNA. Proteins called initiation factors are also required to bring these translation components together (mRNA, large and small ribosomal subunits and initiator tRNA). The ribosome is now fully assembled and ready to begin translation.

Elongation of Translation The assembled ribosome covers about 30 nucleotides of the mRNA. It holds two mRNA codons in alignment with the two tRNA binding sites of the large subunit . A second tRNA, with an anticodon complementary to the second codon of the mRNA, moves into the second tRNA binding site on the large subunit, the A site. The amino acids attached to the two tRNAs are now side by side.

The catalytic site of the large subunit breaks the bond holding the first amino acid (methionine) to its tRNA A peptide bond is formed between these two amino acids After the peptide bond is formed, the first tRNA in the P site which is already ‘empty’ is released and the second tRNA now carries a two-amino acid chain. The tRNA in the A site which carries the two-amino acids chain will translocate to the P site.

A new tRNA, with an anticodon complementary to the third codon of the mRNA, binds to the empty second site (A site). The catalytic site on the large subunit now links the third amino acid onto the growing protein chain. The ‘empty’ tRNA leaves the ribosome, the ribosome shifts to the next codon on the mRNA, and the process repeats, one codon at a time.

Termination of Translation A stop codon in the mRNA molecule signals the ribosome to terminate protein synthesis (3 types of stop codons: UAA, UAG, UGA). Stop codons do not bind to tRNA, but special proteins bind to the ribosome, forcing the ribosome to release the finished protein chain and the mRNA. Newly synthesized polypeptides frequently undergo posttranslational modifications to produce the final active form of protein. During translation, it is usual for several ribosomes to be bound to the same mRNA. Such complex are called a polysome.

An mRNA molecule can be translated in three possible reading frames.

How fast does translation occur?? Can synthesize 5 to 15 peptide bonds per second. Most proteins are 100 to 200 amino acids long ----less than a minute. How fast does translation occur??

MUTATIONS

Point mutations can affect protein structure and function Mutations are changes in the genetic material of a cell (or virus). A chemical change in just one base pair of a gene causes a point mutation. If these occur in gametes or cells producing gametes, they may be transmitted to future generations. For example, sickle-cell disease is caused by a mutation of a single base pair in the gene that codes for one of the polypeptides of hemoglobin. A change in a single nucleotide from T to A in the DNA template leads to an abnormal protein.

A point mutation that results in the replacement of a pair of complementary nucleotides with another nucleotide pair is called a base-pair substitution. Some base-pair substitutions have little or no impact on protein function. In silent mutations, alterations of nucleotides still indicate the same amino acids because of redundancy in the genetic code. Other changes lead to switches from one amino acid to another with similar properties. Still other mutations may occur in a region where the exact amino acid sequence is not essential for function.

Changes in amino acids at crucial sites, especially active sites, are likely to impact function. Missense mutations are those that still code for an amino acid but change the indicated amino acid. Nonsense mutations change an amino acid codon into a stop codon, nearly always leading to a nonfunctional protein. Insertions and deletions are additions or losses of nucleotide pairs in a gene. These have a disastrous effect on the resulting protein more often than substitutions do. Unless these mutations occur in multiples of three, they cause a frameshift mutation.

Mutations can occur in a number of ways. All the nucleotides downstream of the deletion or insertion will be improperly grouped into codons. The result will be extensive missense, ending sooner or later in nonsense - premature termination. Mutations can occur in a number of ways. Errors can occur during DNA replication, DNA repair, or DNA recombination. These can lead to base-pair substitutions, insertions, or deletions, as well as mutations affecting longer stretches of DNA. These are called spontaneous mutations. Spontaneous mutation = A mutation occurring in the absence of mutagens, usually due to errors in the normal functioning of cellular enzymes.

BASE-PAIR SUBSTITUTION

BASE-PAIR INSERTION OR DELETION