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Transcription Mahmoud A. Alfaqih BDS PhD College of Medicine and Health Sciences (CMHS) Department of Medical Biochemistry Arabian Gulf University Central dogma in Biology Transcription is the first step in gene expression The central dogma of biology In Bac...

Transcription Mahmoud A. Alfaqih BDS PhD College of Medicine and Health Sciences (CMHS) Department of Medical Biochemistry Arabian Gulf University Central dogma in Biology Transcription is the first step in gene expression The central dogma of biology In Bacteria and other prokaryotes, translation of mRNA into a polypeptide can take place while mRNA is transcribed from DNA This is called “Transcription – Translation” coupling This is specific to prokaryotes and does not take place in eukaryotic species  RNAP hnRNA Intron Exon DNA 5’ Capping Transcription Splicing Polyadenylation A A A Mature mRNA Polypeptide Chain Transported to cytoplasm A A A Ribosome Translation Transcription Transcription is DNA-directed synthesis of RNA and is the first step in gene expression. Gene expression, is the transformation of a specific nucleotide sequence (genetic information) into a product which influences cellular function. The gene is transcribed into RNA and RNA is either translated into protein (mRNA) or it contributes to translation (rRNA, tRNA) or contributes to the regulation of translation (microRNA). THREE steps in transcription Gene Structure The gene is a minimal linear sequence of nucleotides in DNA which encodes for a protein or structural RNA. The sequence of a gene is written from 5' to 3’. Eukaryotic genes are composed of exons (coding), introns (non-coding) and non-coding consensus (regulatory) sequences. Any nucleotide before the transcription start site has a -ve number (e.g. -1) Any nucleotide located after the start site has a +ve numbers (e.g. +5). Gene Structure Genes producing mRNA have 2 components: Coding region which is transcribed into mRNA, and ultimately translated into protein Regulatory region Basal promoters: a sequence in the gene which ensures basal expression and the initiation of transcription (An example is TATA sequence (known as TATA box) and Other sequences) DNA regulatory elements: regulate gene expression (Examples: Proximal promoter elements, Enhancers and Repressors) Basal promoter CAAT box and GC rich regions are examples of proximal promoter elements While enhancers are an example of distal elements 5’ UTR stands for the 5’ untranslated region. Start codon of translation is not exactly at the transcription Start site Gene Structure Promoter: is a DNA sequence which determines the start site of RNA synthesis (transcription) Promoters are located upstream of the transcription start site. TATA sequence (known as TATA box). Other sequences that may be needed for promoter function are CAAT box & GC box. Basal transcriptional proteins (factors) binds TATA box and link the box to RNA Polymerase II.  Basal Transcription factors In addition to RNA polymerase, basal transcription requires certain transcription factors called A, B, C, D, E, F & H. Many of these factors are also composed of several subunits. These factors are generally described as Transcription factor 2 (letter symbol) e.g. TFIIA. TFIID consists of TATA binding protein (TBP) plus 8 to 10 TBP-associated factors. Synthesis of single stranded RNA from DNA The RNA polymerase is a DNA-dependent RNA polymerase, since it uses the information in the DNA to produce complementary RNA sequence. It uses only one strand of the gene as a template (described as the template strand)(Also known as the non-coding strand). The other DNA strand, known as coding strand, have a similar sequence to the produced RNA, except T is replaced by U in the RNA The RNA polymerase reads the DNA from 3´ to 5´ direction The product is a single strand complementary to the template strand synthesized from 5´ to 3´ direction. Initiation Initiation TATA box Initiator (contains transcription starting site Promotor Elongation TATA box Initiator (contains transcription starting site Promotor Termination hnRNA DNA Termination sequence hnRNA 3’ TATA box Initiator (contains transcription starting site Promotor 5’ Gene- regulatory regions Enhancers and repressor response elements Definition: DNA sequences that regulate gene expression by enhancing (enhancer element) or repressing (repressing element) expression. These sequences mediate either an increase (enhancer) or a decrease (repressor) in the rate of initiation An increase or a decrease in the rate of initiation could be in response to hormones, chemicals….etc The response elements could be found upstream or downstream the initiation site Enhancers or repressors can regulate the expression of more than one gene. Enhancers or repressors can regulate the expression of genes found hundreds or thousands of bps away but on the same chromosome.  RNA synthesis RNA synthesis takes place in the nucleus and is catalyzed by RNA polymerase using DNA as a template. RNA differ from DNA: a. It is single stranded b. Contain Uracil (U) instead of thymine (T) c. Much shorter. Genes which code for proteins are transcribed into mRNA in the nucleus, however, translation takes place in the cytosol. Regulatory sequences in the mRNA (in the 5' & 3' ends, called un-translated region – UTR) are important for the stability and translation efficiency of the mRNA, but are not translated into protein.  RNA Polymerases (Review) RNA polymerase: There are at least 3 distinct RNA polymerases in eukaryotes: RNA polymerase I, catalyze the synthesis of rRNA (28S, 18S, 5.8S) RNA polymerase II, catalyze the synthesis of mRNA and microRNA RNA polymerase III, catalyze the synthesis of tRNA and 5S rRNA Transcription and accessory factors: several proteins, transcription factors and accessory proteins (collectively known as pre-initiation complex) are need for initiation of transcription by proper placing of RNA polymerase II to the DNA (just upstream the start site of the gene). HnRNA (Heterogeneous nuclear RNA) The immediate product of transcription is called primary transcript or heterogeneous nuclear RNA (hnRNA), or pre-mRNA, which is larger than mRNA seen in cytoplasm. The pre-mRNA contain exons (coding sequence) and introns (non-coding) segments. The pre-mRNA undergoes 3 main modifications to give mature mRNA. Addition of 5’ cap Addition of poly-A tail Intron removal (splicing)  Addition of 5’cap Occurs Immediately after initiation of RNA synthesis The cap is a methyl guanosine residue linked via a 5’ to 5’ bridge The cap helps to protect mRNA from degradation by 5´ exonucleases The cap mediates the binding of mRNA with the ribosomes during translation.  Addition of the Poly A tail hnRNA contain a consnensus AAUAAA sequence known as polyadenylation signal several hundreds of nucleotide before the 3´ end. The AAUAAA signal is recognized by a specific endonuclease which cuts the RNA after the signal by 20 nucleotides, the cut part is removed. To the new 3´ end, up to 250 adenine nucleotides are added by poly(A) polymerase enzyme, forming the poly(A) tail.  Polyadenylation Polyadenylation signal 5’ 3’ A A A Recognizes AAUAAA poly(A) polymerase enzyme Endonuclease  splicing - Within the gene there are splice sites which indicate the beginning (GU) and the end (AG) of each intron in the hnRNA. - Introns are removed and exons are spliced (joined) together to form the mature mRNA. - This process is performed by, 5 small nuclear RNAs (snRNA): U1, U2, U5 and U4/6, and more than 50 proteins, collectively known as small nuclear ribonucleo proteins snRNPs (pronounced “snurps”) - The snurps along with the primary transcript (pre-mRNA) form a special structure called spliceosome - The mature mRNA (after splicing) passes through the pores of the nuclear membrane to the cytoplasm - Translation of mRNA occurs in the cytsoplasm.  Splicing Splice acceptor and Splice donor sequences are one type of consensus sequence found at the 3' ends and 5‘ ends of the introns. Introns begin with GU nucleotides (splice donor) and end with AG nucleotides (splice acceptor) preceded by pyrimidine-rich tract. This sequence is essential for splicing (removal) of the introns from the primary transcript. Spliceosome Splice site mutation These are mutations at a splice site which might result in alteration of size and content of the mRNA transcript, resulting in aberrant (abnormal) protein.