Transcription Molecular Biology

IntroductionThe central dogma of molecular biology Gene expression Solid arrows: transfers that occur in all cell Dashed arrows: Special transfers RNA-directed RNA polymerase RNA-directed DNA polymerase (reverse transcriptase) Missing arrows are information transfers never occur: protein specifying either DNA, RNA, or proteinIn other words, proteins can only be the recipients of genetic information

The classical flow of genetic information, often referred to as "The Central Dogma" is:

Transcription The synthesis of an RNA molecule from DNA

A complex process involving one of the group of RNA polymerase enzymes

RNA(n residues) + NTP RNA(n + 1 residues) + PPi

RNA synthesis proceeds in a stepwise manner in the direction, that is, the incoming nucleotide is appended to the free 3-OH group of the growing RNA chainAction of RNA polymerases

RNA Is Synthesized from a DNA Template by an RNA Polymerase The processes of DNA and RNA synthesis are similar in that they involve

(1) the general steps of initiation, elongation, and termination with 5' to 3' polarity; (2) large, multicomponent initiation complexes; and (3) adherence to Watson-Crick base-pairing rules.

These processes differ in several important ways, including the following:

(1) ribonucleotides are used in RNA synthesis rather than deoxyribonucleotides; (2) U replaces T as the complementary base pair for A in RNA; (3) a primer is not involved in RNA synthesis; (4) only a portion of the genome is transcribed or copied into RNA, whereas the entire genome must be copied during DNA replication; (5) there is no proofreading function during RNA transcription.

Watson and Crick model of the double-helical structure of the B form of DNA.

Messenger RNA

The relationship between the sequences of an RNA transcript and its gene, in which the coding and template strands are shown with their polarities.

The RNA transcript with a 5' to 3' polarity is complementary to the template strand with its 3 to 5' polarity. Note that the sequence in the RNA transcript and its polarity is the same as that in the coding strand, except that the U of the transcript replaces the T of the gene.

The Template Strand of DNA Is Transcribed The strand that is transcribed or copied into an RNA molecule is referred to as the template strand of the DNA. The other DNA strand, the non-template strand, is frequently referred to as the coding strand of that gene. It is called this because, with the exception of T for U changes, it corresponds exactly to the sequence of the RNA primary transcript, which encodes the (protein) product of the gene.

In the case of a double-stranded DNA molecule containing many genes, the template strand for each gene will not necessarily be the same strand of the DNA double helix Thus, a given strand of a double-stranded DNA molecule will serve as the template strand for some genes and the coding strand of other genes.

Note that the template strand is always read in the 3' to 5' direction.

The Prokaryotic Transcription DNA-Dependent RNA Polymerase Initiates Transcription at a Distinct Site, the Promoter E coli RNAP consists of a core complex of : 2, often termed E, associates with a spesific protein factor (sigma factor) to form holoenzyme, 2 or E

The transcription "bubble" is an approximately 20-bp area of melted DNA, and the entire complex covers 3075 bp, depending on the conformation of RNAP.

The transcription cycle in bacteria

Function of the transcription bubble RNA polymerase unwinds the DNA double helix, it separates a short segmet (10 bp) to form transcription bubble, thereby permitting this portion to transiently form a short DNARNA hybrid helix.

Eukaryotic cells contain five different types of RNA polymerases that each synthesize a different class of RNA. In bacteria, one species of this enzyme synthesizes all of the cells RNA.

Bacterial RNA transcription is described in five steps:

(1) Template binding: RNAP binds to DNA and locates a promoter (P) melts the two DNA strands to form a preinitiation complex (PIC).

(2) Chain initiation: RNAP holoenzyme (core + one of multiple factors) catalyzes the coupling of the first base (usually ATP or GTP) to a second ribonucleoside triphosphate to form a dinucleotide.

(3) Promoter clearance: RNAP undergoes a conformational change after RNA chain length reaches 1020 nt and then is able to move away from the promoter, transcribing down the transcription unit.

(4) Chain elongation: Successive residues are added to the 3'-OH terminus of the nascent RNA molecule.

(5) Chain termination and release: The completed RNA chain and RNAP are released from the template. The RNAP holoenzyme re-forms, finds a promoter, and the cycle is repeated.

Bacterial promoters, E Coli The Fidelity & Frequency of Transcription Is Controlled by Proteins Bound to Certain DNA Sequences.

Bacterial Promoters Are Relatively Simple TATA box (Pribnow-Schaller box )

Specific proteins known in prokaryotes as activators and repressors and in eukaryotes as transcription factors bind to these control sites (promoter) Transcriptional Termination Is a Relatively Simple Process

Rho-dependent transcription termination signals Bacterial transcription termination signal contains: inverted repeat ( dyad symetry) (the two boxed areas) followed by a stretch of AT base pairs.

The inverted repeat, when transcribed into RNA, can generate the hairpin secondary structure in the RNA transcript. Formation of this RNA hairpin causes RNAP to pause and subsequently the termination factor interacts with the paused polymerase and somehow induces chain termination.

Transcription continues into the AT region, and with the aid of the termination protein the RNA polymerase stops, dissociates from the DNA template, and releases the nascent transcript.

Rho-dependent transcription termination signals Bacterial transcription termination signal contains: inverted repeat ( dyad symetry) (the two boxed areas) followed by a stretch of AT base pairs.

The inverted repeat, when transcribed into RNA, can generate the hairpin secondary structure in the RNA transcript. Formation of this RNA hairpin causes RNAP to pause and subsequently the termination factor interacts with the paused polymerase and somehow induces chain termination.

Transcription continues into the AT region, and with the aid of the termination protein the RNA polymerase stops, dissociates from the DNA template, and releases the nascent transcript.

Eukaryotic Promoters Are More Complex

Proximal and distal cis elements are bound by trans -acting transcription factors, in this example: Sp1 and CTF (also called C/EBP, NF1, NFY). These cis elements can function independently of orientation (arrows).

Schematic diagram showing the transcription control regions in a hypothetical mRNA-producing, eukaryotic gene transcribed by RNA polymerase II. Such a gene can be divided into its coding and regulatory regions, as defined by the transcription start site (arrow; +1).

Basal Transcription Complex Formation of the basal transcription complex begins when TFIID binds to the TATA box. It directs the assembly of several other components by protein-DNA and protein-protein interactions; TFIIA, B, E,F, H, and polymerase II (pol II). The entire complex spans DNA from position 30 to +30 relative to the initiation site (+1, marked by bent arrow)

Nucleosome eviction by chromatin-active coregulators facilitates PIC formation and transcription.Promoter Accessibility and Hence PIC Formation Is Often Modulated by Nucleosomes

Transcription Factors

Two Models Can Explain the Assembly of the Preinitiation Complex

A. Stepwise B. Holoenzyme TFIID, TFIIA, TFIIB, TFIIE, TFIIH, TFIIF, Med Binding of a single protein complex: pol II, Med and six GTFs

RNA MOLECULES ARE USUALLY PROCESSED BEFORE THEY BECOME FUNCTIONAL

The Coding Portions (Exons) of Most Eukaryotic Genes Are Interrupted by Introns

Introns Are Removed & Exons Are Spliced Together

Alternative Splicing Provides for Different mRNA

Alternative Promoter Utilization Provides a Form of Regulation

Messenger RNA (mRNA) Is Modified at the 5' & 3' Ends

The classical flow of genetic information, often referred to as "The Central Dogma" is:

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