Expression of the genome. Books: 1. Molecular biology of the gene: Watson et al 2. Genetics: Peter J. Russell

Transcription

1 Expression of the genome Books: 1. Molecular biology of the gene: Watson et al 2. Genetics: Peter J. Russell 1

2 Transcription 1. Francis Crick (1956) named the flow of information from DNA RNA protein the Central Dogma. 2. Synthesis of an RNA molecule using a DNA template is called transcription. Only one of the DNA strands is transcribed. The enzyme used is RNA polymerase. 3. There are four major types of RNA molecules: a. Messenger RNA (mrna) encodes the amino acid sequence of a polypeptide. b. Transfer RNA (trna) brings amino acids to ribosomes during translation. c. Ribosomal RNA (rrna) combines with proteins to form a ribosome, the catalyst for translation. d. Small nuclear RNA (snrna): minor class; combines with proteins to form complexes used in eukaryotic RNA processing. 2

3 The Transcription Process RNA Synthesis 1. Transcription, or gene expression, is regulated by gene regulatory elements associated with each gene. 2. DNA unwinds in the region next to the gene, due to RNA polymerase in prokaryotes and other proteins in eukaryotes. In both, RNA polymerase catalyzes transcription (Figure 5.1). 3

4 Fig. 5.1 Transcription process 4 Peter J. Russell, igenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.

5 3. RNA is transcribed 5 -to-3. The template DNA strand is read 3 -to-5. Its complementary DNA, the nontemplate strand, has the same polarity as the RNA. 4. RNA polymerization is similar to DNA synthesis (Figure 5.2), except: a. The precursors are NTPs (not dntps). b. No primer is needed to initiate synthesis. c. No proofreading occurs. d. Uracil is inserted instead of thymine. 5

6 Fig. 5.2 Chemical reaction involved in the RNA polymerasecatalyzed synthesis of RNA on a DNA template strand 6 Peter J. Russell, igenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.

7 The Transcription Process Initiation of Transcription at Promoters 1. Transcription is divided into three steps for both prokaryotes and eukaryotes. They are initiation, elongation and termination. The process of elongation is highly conserved between prokaryotes and eukaryotes, but initiation and termination are somewhat different. 2. This section is about initiation of transcription in prokaryotes. E. coli is the model organism. 3. A prokaryotic gene is a DNA sequence in the chromosome. The gene has three regions, each with a function in transcription: a. A promoter sequence that attracts RNA polymerase to begin transcription at a site specified by the promoter. b. The transcribed sequence, called the RNA-coding sequence. The sequence of this DNA corresponds with the RNA sequence of the transcript. c. A terminator region downstream of the RNA-coding sequence that specifies where transcription will stop. 7

8 Fig. 5.3 Promoter, RNA-coding sequence, and terminator regions of a gene 8 Peter J. Russell, igenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.

9 4. Promoters in E. coli generally involve two DNA sequences, centered at -35 bp and -10 bp upstream from the +1 start site of transcription. 5. The common E. coli promoter that is used for most transcription has these consensus sequences: a. For the -35 region the consensus is 5 -TTGACA-3. b. For the -10 region (previously known as a Pribnow box), the consensus is 5 -TATAAT-3. Promoters often deviate from consensus. The associated genes will show different levels of transcription, corresponding with sigma s ability to recognize their sequences. 6. Transcription initiation requires the RNA polymerase holoenzyme to bind to the promoter DNA sequence. Holoenzyme consists of: a. Core enzyme of RNA polymerase, containing four polypeptides (two, one and one ). b. Sigma factor ( ). 7. Sigma factor binds the core enzyme, and confers ability to recognize promoters and initiate RNA synthesis. Without sigma, the core enzyme randomly binds DNA but does not transcribe it efficiently. 8. RNA polymerase holoenzyme binds promoter via the sigma factor in two steps (Figure 5.4): a. First, it loosely binds to the -35 sequence. b. Second, it binds tightly to the -10 sequence, untwisting about 17 bp of DNA at the site, and in position to begin transcription. 9

10 Sigma factor A sigma factor (σ factor) is a prokaryotic transcription initiation factor that enables specific binding of RNA polymerase to gene promoters. Different sigma factors are activated in response to different environmental conditions. Every molecule of RNA polymerase contains exactly one sigma factor subunit. E. coli has seven sigma factors; the number of sigma factors varies between bacterial species. Sigma factors are distinguished by their characteristic molecular weights. For example, σ70 refers to the sigma factor with a molecular weight of 70 kda. σ70 (RpoD) - the "housekeeping" sigma factor or also called as primary sigma factor, transcribes most genes in growing cells. Makes the proteins necessary to keep the cell alive. 10

11 Fig. 5.4 Action of E. coli RNA polymerase in the initiation and elongation stages of transcription 11 Peter J. Russell, igenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.

12 The Transcription Process Elongation and Termination of an RNA Chain 1. Once initiation is completed, RNA synthesis begins. After 8 9 NTPs have been joined in the growing RNA chain, sigma factor is released and reused for other initiations. Core enzyme completes the transcript (Figure 5.4). 2. Core enzyme untwists DNA helix locally, allowing a small region to denature. Newly synthesized RNA forms an RNA-DNA hybrid, but most of the transcript is displaced as the DNA helix reforms. The chain grows at nt/second. 12

13 3. Terminator sequences are used to end transcription. In prokaryotes there are two types: a. Rho-independent ( -independent) or type I terminators have two-fold symmetry that would allow a hairpin loop to form (Figure 5.5). The palindrome is followed by 4-8U residues in the transcript, and together these sequences cause termination, possibly because rapid hairpin formation destabilizes the RNA-DNA hybrid. b. Rho-dependent ( -dependent) or type II terminators lack the poly(u) region, and many also lack the palindrome. The protein is required for termination. Forms a hexameric ring It has two domains, one binding RNA and the other binding ATP. ATP hydrolysis provides energy for to move along the transcript and destablize the RNA-DNA hybrid at the termination region. Rho binds to rut sites in RNA (Rho-utilization sites); 40 nucleotides that remain single-stranded; rich in C residues. Rho does not bind a transcript being translated by ribosomes. Rho-dependent terminators account for about half of E. coli terminators. The other terminators discovered for E. coli are called Tau and nusa. Rho-dependent terminators were first discovered in bacteriophage genomes. 13

14 Fig. 5.5 Sequence of a -independent terminator and structure of the terminated RNA 14 Peter J. Russell, igenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.

15 Rho-dependent Rho-independent 15