Gene Expression: Transcription, Translation, RNAs and the Genetic Code

Transcription

1 Lecture Gene Expression: Transcription, Translation, RNAs and the Genetic Code Central dogma of molecular biology During transcription, the information in a DNA sequence (a gene) is copied into a complementary RNA sequence. During translation, this RNA sequence is used to create the amino acid sequence of a polypeptide. RNA (ribonucleic acid) differs from DNA and plays a vital role in gene expression RNA generally consists of only one polynucleotide strand composed of nucleotides The sugar molecule found in RNA is ribose, rather than the deoxyribose found in DNA. Three of the nitrogenous bases (adenine, guanine, and cytosine) in RNA are identical to those in DNA, the fourth base in RNA is uracil (U), which is similar to thymine but lacks the methyl ( CH3) group. Types of RNA Messenger RNA (mrna) carries a copy of a gene sequence in DNA to the site of protein synthesis at the ribosome. Formed during transcription from a DNA template. Transfer RNA (trna) carries amino acids to the ribosome for assembly into polypeptides. Anticodon matches with codon of mrna. Specific amino acid for each anticodon attached to 3 end of trna. Ribosomal RNA (rrna) catalyses peptide bond formation and provides a structural framework for the ribosome. Micro RNA (mirna) is single stranded non-specific RNA that can influence many mrna sequences, produced by specific genes, and inhibit translation of mrna. A type of interference RNA. Small interfering RNA (sirna) is double stranded and a highly specific RNA that targets another RNA sequence. Cleaves mrna so it is degraded and cannot be translated Transcription Initiation: RNA polymerase binds to the promoter and starts to unwind the DNA strands. Elongation: RNA polymerase moves along the DNA template strand from 3 to 5 and produces the RNA transcript by adding nucleotides to the 3 end of the growing RNA. Termination: When RNA polymerase reaches the termination site, the RNA transcript is set free from the template. Naming of strands Template strand is transcribed into a complementary mrna sequence but the sequence on the mrna is similar to the non-template strand except U instead of A. The non-template strand is called

2 the coding strand and is often used when showing a sequence because it represents the mrna sequence INITIATION Initiation requires a promoter, a special sequence of DNA to which the RNA polymerase binds very tightly. Eukaryotic genes generally have one promoter each, while in prokaryotes and viruses, several genes often share one promoter. Promoters are important control sequences that tell the RNA polymerase where to start transcription and which strand of DNA to transcribe. DNA melts (open strands) in region of binding and transcription can begin RNA polymerases catalyse the synthesis of RNA from the DNA template. Prokaryotes: All genes transcribed by a single RNA polymerase with associated detachable subunit the sigma factor (σ). Core enzyme composed of 5 subunits α2ββω and the sixth unit is σ; when bound it is called the holoenzyme. Core enzyme + σ Holoenzyme 1. The sigma factor of RNA polymerase recognises and binds to promoter regions forming closed promoter complex and opens double strands of DNA. 2. Once core enzyme is bound the sigma factor dissociates from the rest of the complex 3. Promoters are usually 10 or 35 bases upstream from starting point of transcription. Eukaryotes: Eukaryotes have three kinds of promoters. Promoters contain sequences that determine specificity of RNA polymerase binding. Most promoters have regulatory elements, a TATA box and a transcription start site. RNA polymerase I: rrna genes RNA polymerase II: protein encoding genes RNA polymerase III: trna, snrna RNA polymerase II cannot recognise promoters on their own and require general transcription factors which assemble at the promoter which the polymerase then binds onto to start transcription Binding of repressor protein can block transcription Binding of activator protein stimulates transcription

3 Elongation RNA polymerase unwinds the DNA about 10 base pairs at a time and reads the template strand in the 3 -to-5 direction. RNA polymerase adds new nucleotides to the 3 end of the growing strand, but does not require a primer to get this process started. The new RNA elongates from the first base, which forms its 5 end, to its 3 end. Termination RNA polymerase reaches a chain termination sequence which can form a loop when transcribed Termination sequence contains multiple G and C so it is termed GC hairpin RNA polymerase and mrna are released RNA processing (pre-rna, primary transcript, hnrna into mrna) Eukaryotes must edit and be modified before it leaves nucleus G cap is added to the 5 end of the pre-mrna as it is transcribed. The G cap is a chemically modified molecule of guanosine triphosphate (GTP). It facilitates the binding of mrna to the ribosome for translation, and it protects the mrna from being digested by ribonucleases that break down RNAs. Poly A tail is added to the 3 end of the pre-mrna at the end of transcription. A polyadenylation sequence (AAUAAA) near the 3 end of the pre-mrna, after the last codon which acts as a signal for an enzyme to cut the pre-mrna. Immediately after this cleavage, another enzyme adds 100 to 300 adenine nucleotides (a poly A sequence) to the 3 end of the pre-mrna. This tail may assist in the export of the mrna from the nucleus and is important for mrna stability. RNA splicing removes the introns and splices the exons together. Using energy from ATP, proteins are added to form a large RNA protein complex called a spliceosome. This complex cuts the pre-mrna, releases the introns, and joins the ends of the exons together to produce mature mrna. Introns are non-coding base sequences which are removed from pre-mrna Exons are the base sequence which are expressed Alternative splicing results in different mature mrnas and proteins

4 Genetic Code Genetic code relates genes (DNA) to mrna and mrna to the amino acids that make up proteins. The genetic code specifies which amino acids will be used to build a protein. Genetic information is encoded in mrna in three-letter units codons made up of nucleoside monophosphates with the bases uracil (U), cytosine (C), adenine (A), and guanine (G) and is read in a 5 to 3 direction on mrna. AUG, which codes for methionine, is also the start codon, the initiation signal for translation. Three of the codons (UAA, UAG, and UGA) are stop codons, or termination signals for translation. When the translation machinery reaches one of these codons, translation stops, and the polypeptide is released from the translation complex.

5 Genetic code is degenerate and there is more than one codon for some amino acids. i.e. synonymous codons Genetic code is non-overlapping and the code starts from a fixed point Transfer RNAs carry specific amino acids and bind to specific codons trna binds to a particular amino acid. When it is carrying an amino acid, the trna is said to be charged. It associates with mrna and interacts with ribosomes. The 3 end of every trna molecule is its amino acid attachment site: a site to which its specific amino acid binds covalently. The midpoint of the trna sequence is a group of three bases, called the anticodon, which is the site of complementary base pairing (via hydrogen bonding) with the codon on the mrna. Thus, each trna species has a unique anticodon that corresponds to the amino acid it carries. Activating enzymes link the right trnas and amino acids trna + amino acid = aminoacyl-trna

6 Ribosomes Where the task of translation is accomplished. Its structure enables it to hold mrna and charged trnas in the right positions, thus allowing a polypeptide chain to be assembled efficiently. Each ribosome consists of two subunits, a large one and a small one On the large subunit of the ribosome there are three sites to which a trna can bind to The A (amino acid) site is where the charged trna anticodon binds to the mrna codon, thus lining up the correct amino acid to be added to the growing polypeptide chain. The P (polypeptide) site is where the trna adds its amino acid to the polypeptide chain. The E (exit) site is where the trna, having given up its amino acid, resides before being released from the ribosome and going back to the cytosol to pick up another amino acid and begin the process again Translation Initiation The small ribosomal subunit binds to its recognition sequence on mrna. Prokaryotes: Small ribosome unit (30S) and mrna bind at Shine Delgarno sequence (ribosome binding site) at the 5 untranslated region Eukaryotes: Initiation factors associated with 5 cap, small ribosomal unit (40S) and Met trna search for start codon. Methionine-charged trna binds to the AUG start codon, completing the initiation complex. The large ribosomal subunit joins the initiation complex, with methionine charged trna now occupying the P site. Elongation Codon recognition: The anticodon of an incoming trna binds to the codon at the A site. Peptide bond formation: Second amino acid is linked to Met by peptidyl transferase activity of the large subunit. Elongation: Free trna is moved to the E site, and then released (uncharged), as the ribosome shifts by one codon, so that the growing polypeptide chain moves to the P site Termination A release factor binds to the complex when a stop codon enters the A site. The release factor disconnects the polypeptide from the trna in the P site. The remaining component s (mrna and ribosomal subunits) separate.

7 Gene Regulation Chromatin Structure Transcriptional environmental influence, enhancers and repressors Post translational