Recall Some points of Replication

DR. ABID ALI DEPARTMENT OF BIOCHEMISTRY DNA TRANSCRIPTION DR. ABID ALI DEPARTMENT OF BIOCHEMISTRY

Learning Objectives Students will be able to Discuss DNA Transcription Explain Post transcriptional Modifications

DNA directed RNA synthesis Transcription DNA directed RNA synthesis

Basic structure of a gene Regulatory region coding region

Transcription Transcription is the mechanism by which a template strand of DNA is utilized by specific RNA polymerases to generate one of the three different types of RNA.

Types of RNA 1) Messenger RNA (mRNA) This class of RNAs are the genetic coding templates used by the translational machinery to determine the order of amino acids incorporated into an elongating polypeptide in the process of translation.

mRNA If the mRNA carries information from more than one gene. It is said to be polycistronic (cistron=gene). This is the characteristic of Prokaryotes If the mRNA carries information from just one gene. It is said to be monocistronic and is characteristic of eukaryotes.

Types of RNA….. 2) Transfer RNA (tRNA) This class of small RNAs form covalent attachments to individual amino acids and recognize the encoded sequences of the mRNAs to allow correct insertion of amino acids into the elongating polypeptide chain.

Types of RNA….. 3) Ribosomal RNA (rRNA) This class of RNAs are assembled, together with numerous ribosomal proteins, to form the ribosomes. Ribosomes engage the mRNAs and form a catalytic domain into which the tRNAs enter with their attached amino acids. The proteins of the ribosomes catalyze all of the functions of polypeptide synthesis

Where does transcription take place?

Transcription in eukaryotes Step 1: transcribing a primary RNA transcript Step 2: modification of this transcript into mRNA

Steps in RNA Synthesis The process of transcription of a typical gene can be divided into three phases: initiation, elongation, and termination. A transcription unit extends from the promoter to the termination region, and the initial product of transcription by RNA polymerase is termed the primary transcript.

Step 1 - overview Initiation Elongation C. Termination A) RNA polymerase binds to promoter region & opens helix Chain elongation in 5’-3’ direction C) stops at termination signal

Initiation: SIGNAL specific DNA sequences called promoters 1) Region where RNA polymerase binds to initiate transcription 2) Sequence of promoter determines direction of RNA polymerase action 3) Rate of gene transcription depends on rate of formation of stable initiation complexes

A) Initiation: ENZYME RNA polymerase holoenzyme Transcription begins with the binding of the RNA polymerase holoenzyme to a region of the DNA known as the promoter, The prokaryotic promoter contains consensus sequences are idealized sequences in which the base shown at each position is the base most frequently encountered at that position.

–35 sequence: A consensus sequence (5'-TTGACA-3'), centered about 35 bases to the left of the transcription start site (see Figure), is the initial point of contact for the holo enzyme. The regulatory sequences that control transcription are, by convention, designated by the 5'→3' nucleotide sequence on the coding strand.

Pribnow box The holoenzyme moves and covers a second consensus sequence (5'-TATAAT-3'), centered at about –10 (see Figure ), which is the site of initial DNA melting (unwinding). Melting of a short stretch (about 14 bases) converts the closed complex to an open one known as a transcription bubble.

Elongation Once the promoter region has been recognized and bound by the holoenzyme, local unwinding of the DNA helix continues (Figure), mediated by the polymerase . [Note: Unwinding generates supercoils in the DNA that can be relieved by DNA topoisomerases.]

Elongation RNA polymerase begins to synthesize a transcript of the DNA sequence, and several short pieces of RNA are made and discarded. The elongation phase is said to begin when the transcript (typically starting with a purine) exceeds ten nucleotides in length. Sigma is then released, and the core enzyme is able to leave (“clear”) the promoter and move along the template strand in a processive manner.

During transcription, a short DNA-RNA hybrid helix is formed (see Figure 30.8). Like DNA polymerase , RNA polymerase uses nucleoside triphosphates as substrates and releases pyrophosphate each time a nucleoside monophosphate is added to the growing chain. As with replication, transcription is always in the 5'→3' direction. In contrast to DNA polymerase , RNA polymerase does not require a primer and does not appear to have proofreading activity.

Transcription stops when RNA polymerase reaches a section of DNA called the terminator Terminator sequence = AAUAAA Next, the RNA strand is released and RNA polymerase dissociates from the DNA The RNA strand will go through more processing

Termination The elongation of the single-stranded RNA chain continues until a termination signal is reached. Termination can be intrinsic (spontaneous) or dependent upon the participation of a protein known as the ρ (rho) factor.

ρ-Independent termination Seen with most prokaryotic genes, this requires that a sequence in the DNA template generate a sequence in the nascent (newly made) RNA that is self-complementary (Figure 30.9). This allows the RNA to fold back on itself, forming a GC-rich stem (stabilized by H-bonds) plus a loop. This structure is known as a “hairpin”. Additionally, just beyond the hairpin, the RNA transcript contains a string of Us at the 3'-end.

The bonding of these Us to the complementary As of the DNA template is weak. This facilitates the separation of the newly synthesized RNA from its DNA template, as the double helix “zips up” behind the RNA

ρ-Dependent termination This requires the participation of an additional protein, rho (ρ), which is a hexameric adenosine triphosphatase ( ATPase ) with helicase activity. ρ binds a C-rich “rho recognition site” near the 3'-end of the nascent RNA and, using its ATPase activity, moves along the RNA until it reaches the RNA polymerase paused at the termination site. The ATPdependent helicase activity of ρ separates the RNA-DNA hybrid helix, causing the release of the RNA.

Transcription of Eukaryotic Genes The transcription of eukaryotic genes is a far more complicated process than transcription in prokaryotes. Eukaryotic transcription involves separate polymerases for the synthesis of rRNA, tRNA, and mRNA. In addition, a large number of proteins called transcription factors (TFs) are involved. TFs bind to distinct sites on the DNA—either within the core promoter region, close (proximal) to it, or some distance away (distal).

They are required both for the assembly of a transcription complex at the promoter and the determination of which genes are to be transcribed. [Note: Each eukaryotic RNA polymerase has its own promoters and TFs.] For TFs to recognize and bind to their specific DNA sequences, the chromatin structure in that region must be altered (remodeled) to allow access to the DNA.

Chromatin Structure A major mechanism by which chromatin is remodeled is through acetylation of lysine residues at the amino terminus of histone proteins (Figure 30.11). Acetylation, mediated by histone acetyltransferases ( HATs ) , eliminates the positive charge on the lysine and thereby decreases the interaction of the histone with the negatively charged DNA. Removal of the acetyl group by histone deacetylases ( HDACs ) restores the positive charge, and fosters stronger interactions between histones and DNA.

Nuclear RNA polymerases of eukaryotes There are three distinct classes of RNA polymerase in the nucleus of eukaryotic cells. All are large enzymes with multiple subunits. Each class of RNA polymerase recognizes particular types of genes.

1. RNA polymerase I: This enzyme synthesizes the precursor of the 28S, 18S, and 5.8S rRNA in the nucleolus. 2. RNA polymerase II: This enzyme synthesizes the nuclear precursors of mRNA that are subsequently translated to produce proteins. 3. RNA polymerase III: This enzyme synthesizes tRNA, 5S rRNA, and some snRNA and snoRNA.

Promoters for RNA pol II In some genes transcribed by RNA polymerase II , a sequence of nucleotides that is nearly identical to that of the Pribnow box This promoter consensus sequence is called the TATA or Hogness box. In other genes, for example, those that are always (“constitutively”) expressed, no TATA box is typically present. Instead, a GC-rich region (GC box) may be found.

PROMOTERS Eukaryotes Near 5’ end of genes Recognised by RNA pol II Consensus promoter sequence for constitutive structural genes – GGGCGG Selective structural genes – TATA

ENHANCERS Sequences that are associated with a promoter Enhance the activity of a promoter due to its association with proteins called transcription factors Enhancer contain DNA sequences called response elements that binds with trnscriptional activator. Enhancers mediate most selective gene expression in eukaryotes

Step 2: Modification Post transcriptional processing 3 main steps RNA capping, polyadenylation splicing

Post transcriptional processing Control of gene expression following transcription but before translation Conversion of primary transcript into mature mRNA Occurs primarily in eukaryotes Localised in nucleus

Post transcriptional processing

1) Capping Addition of 7 methylguanosine at 5’ end Mediated by guanylyltransferase Probably protects against degradation Serves as recognition site for ribosomes Transports hnRNA from nucleus to cytoplasm

2) Tailing Addition of poly(A) residues at 3’ end of RNA Transcript cleaved 15-20nt past AAUAAA Poly(A)polymerase and cleavage & polyadenylation specificity factor (CPSF) attach poly(A) generated from ATP These tails help stabilize the mRNA, facilitate its exit from nucleus and aid in translation.

3) Splicing Highly precise removal of intron sequences Performed by spliceosomes (large RNA-protein complex made of small nuclear ribonucleoproteins) Recognise exon-intron boundaries and splice exons together by transesterification reactions

Splicing

Practice Questions

1. The enzyme which builds a mRNA strand complimentary to the DNA transcription unit is called: a. DNA polymerase b. RNA polymerase c. Helicase d. DNA ligase

Key: B a. DNA polymerase: Main polymerizing enzyme of DNA replication. b. RNA polymerase: Main polymerizing enzyme of RNA transcription. c. Helicase: It unwind the DNA d. DNA ligase is an enzyme that repairs breaks in the backbone of double-stranded DNA molecules

2. Proteins that assist the binding of RNA polymerase in prokaryotes to the promoter region on DNA strand are called:- a. TATA binding protein b. SSB proteins c. Sigma factors d. Rho factors

Key: C a. The TATA-binding protein (TBP) is a general transcription factor in eukaryotes.  b. SSB proteins: Single-stranded DNA is produced during all aspects of DNA metabolism: replication, recombination, and repair. c. Sigma factors: A sigma factor (σ factor) is a protein needed only for initiation of transcription d. Rho factors: A ρ factor (Rho factor) is a prokaryotic protein involved in the termination of transcription.

3 RNA produced from a fragment of DNA has the sequence of AAUUGGCU 3 RNA produced from a fragment of DNA has the sequence of AAUUGGCU. The sequence of the corresponding non template strand in the DNA will be:- a. AGCCAATT b. AAUUGGCU c. AATTGGCT d. TTAACCGA

Key: C RNA produced from a fragment of DNA has the non template sequence of AAUUGGCU is replacing U to T. AATTGGCT

4. The promoter sequence in eukaryotes is:- a. TATAAT b. TATAAA c 4. The promoter sequence in eukaryotes is:- a.TATAAT b. TATAAA c. TTGACA d. GTTAAA

Key: B TATAAA The TATA box is a DNA sequence determined in the promoter region of eukaryotes.

5. RNA forms a hairpin structure which displaces RNA Polymerase to stop transcription is called as: Rho dependent Rho independent Terminating factor Sigma factor

Key: B Intrinsic (or rho-independent) termination is when the RNA forms a hairpin structure which displaces RNA Polymerase and stops transcription.  Rho-dependenttermination occurs when the rho protein disassociates the RNA Polymerase and moves it off of the template