Books: 1. Molecular biology of the gene: Watson et …anisam2.yolasite.com/resources/10nov10lecture13.pdf1. Molecular biology of the gene: Watson et al 2. Genetics: Peter J. Russell

1

Books:1.

Molecular biology of the gene: Watson et al

2.

Genetics: Peter J. Russell

Expression of the genome

2

Transcription• 1. Francis Crick (1956) named the flow of information from

DNA RNAprotein

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.

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).

4Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

Fig. 5.1 Transcription process

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.

6Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

Fig. 5.2 Chemical reaction involved in the RNA polymerase- catalyzed synthesis of

RNA on a DNA template strand

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.

8Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

Fig. 5.3 Promoter, RNA-coding sequence, and terminator regions of a gene

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 α, one β and one β’).

–

b. Sigma factor (σ).•

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.

10

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.

Sigma factor

11Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

Fig. 5.4 Action of E. coli RNA polymerase in the initiation and elongation stages of

transcription

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 30–50 nt/second.

13

3. Terminator sequences are used to end transcription. In prokaryotes there are two types:

a. Rho-independent (ρ-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 (ρ-dependent) or type II terminators lack the poly(U) region, and many also lack the palindrome. The protein ρ 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 ρ 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.

14Peter J. Russell, iGenetics: Copyright © Pearson Education, Inc., publishing as Benjamin Cummings.

Fig. 5.5 Sequence of a -independent terminator and structure of the terminated RNA

15

Rho-independent

Rho-dependent