KUFA MEDICAL COLLEGE MGD MODULE SESSION 5: LECTURE 9 MARCH 16, 2014 DR.THEKRA AL-KASHWAN.
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Transcript of KUFA MEDICAL COLLEGE MGD MODULE SESSION 5: LECTURE 9 MARCH 16, 2014 DR.THEKRA AL-KASHWAN.
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KUFA MEDICAL COLLEGE
MGD MODULE
SESSION 5:LECTURE 9 MARCH 16, 2014
DR.THEKRA AL-KASHWAN
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WHAT IS A GENE AND
TRANSCRIPTION
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At the end of this lecture you should be able to:
Describe the process and role of transcription. (LO 7.1)
Define the term gene. (LO 7.3)
List and summarize the major reactions involved the process of RNA
maturation in eukaryotes and explain their importance in gene expression.
(LO 7.4)
Contrast the different types of RNA molecule, i.e. mRNA, rRNA and
tRNA. (LO 7.7)
Compare and contrast gene expression in mammalian and bacterial cells
and explain how the differences can be exploited clinically. (LO 7.8)
Predict the effects of various mutations in a gene. (LO 7.9)
Explain how mutations outside the coding region can affect gene
expression. (LO 7.10)
Intended learning outcomes
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The Central Dogma: is The flow
information from DNA to RNA to Protein
in all organism, with exception of Some
viruses have RNA as the repository of their
genetic information.
The Central Dogma of Molecular biology
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Gene expression: is the process by which the genetic code (the nucleotide sequence) of a gene is used to direct protein synthesis and produce the structures of the cell.
Gene expression involves two main stages:1-Transcription: Transfer of genetic information from the base sequence of DNA to the base sequence of RNA, mediated by RNA synthesis that occur at nucleus.2-Translation: Conversion of information encoded in the nucleotide sequence of an mRNA molecule into the linear sequence of amino acids in a protein that occur at cytoplasm.
Gene expression
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The gene: is the basic physical and functional unit of heredity.
It consists of a specific sequence of nucleotides at a given
position on a given chromosome that codes for a specific
protein (or for an RNA molecule).
Human carrying between 20000-25000 genes that encoded for
all the proteins. These protein–coding genes make up 1–2% of
the human genome and transcribed into mRNA.
Some other genes produce of other forms of RNA: including
transfer RNA (tRNA) and ribosomal RNA (rRNA) involved in
Translation.
What is the Gene?
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Each cell in our body has the same protein –coding genes (the
same genotype) but not all these genes are expressed in every cell.
In fact, in a given cell, almost all genes are switched off most of
the time and only about 5% to 10% of the genes in most cells are
active.
Liver cells, for example, do not express the genes for eye color,
and brain cells do not make enzymes that help digest food.
The process of turning genes on and off is called gene regulation.
So, different cell types use different genes to expresses different
proteins (different phenotype ) making them to differ from each
other.
Gene regulation: regulate the gene expression
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RNA: is polymer of ribonucleotides covalently linked by 3' →5' phosphodiester
RNA is single strand that has direction from 5 ' 3‘ and Bases sequence always written from 5'-end to 3'-end
5'- AGCU-3' Phosphodiester bonds can be cleaved hydrolytically by
chemicals, or hydrolyzed enzymatically by nucleases (ribonucleases): Endonucleases cut the sugar-phosphate backbone
within the sequence, either non-specifically or in a sequence-specific manner (ie at a particular site or sites along the strand).
Exonucleases remove one nucleotide at a time from the ends of the molecule, either in a 5’-specific manner or from the 3’ end.
Review the RNA structure
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A typical eukaryotic gene consists of the following regions:
1-Transcribed region: involved exons and introns
This region contains the DNA sequence that is transcribed into
mRNA
2-Regulatory regions (Gene control regions): involved
Promoter and enhancer and response elements
These regions regulate the transcription of gene
Gene Structure
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1-Transcribed region:Exons: is characterized by the following:
• Code for amino acids and collectively determine the amino acid sequence of the protein product
• Present in final mature mRNA molecule
• Numbered from 5'-end of the gene: exon 1, exon 2, etc. • Exon 1 at 5'-end of the gene has Untranslated region (5'UTR) and coding
region that began with initiation codon (ATG) specify methionine.• Last exon at 3'-end of the gene has coding region ends with stop
codon(TAA,TAG,TGA) that do not specify any amino acid and Untranslated region (3'UTR) .
• 5'UTR is leader of mRNA strand and 3'UTR is tailing. • Mutations in the exons may usually lead to abnormal protein.
Gene Structure
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1-Transcribed region:
Introns: is characterized by the following
• Do not code for amino acids
• Removed (spliced) from the mature mRNA • Each intron always began and ends with consensus sequence:
GT at 5'-end (5'splice donor) and AG at 3'-end (3'splice acceptor). These are essential for splicing introns out of the primary transcript• Mutation at splice sites result in loss of gene production
Gene Structure
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1-Transcribed region: This region start with the base (py A py) serve as start site (startpoint) for transcription, numbered +1, which is first nucleotide incorporated into the RNA at the 5'-end of the transcript.
Subsequent nucleotides in the transcribed region are numbered +2, +3, etc., the direction is called downstream.
Regulatory region of the gene, are numbered –1, –2, –3, etc. from the startpoint, the direction is called upstream.
Gene Structure
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2-Regulatory regions : Promoter
Consisting of a few hundred nucleotides 'upstream' of the
gene (toward the 5' end) that plays a role in controlling the
transcription of the gene: determine the startpoint and
frequency of transcription by controlling the binding RNA
polymerase II
Gene Structure
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2-Regulatory regions : Promoter
Has Consensus sequences represent in:1) TATA box: TATA(A/T)A located -25 region Binds with general
transcriptional factors and directs the RNA polymerase II to the correct site (start site) and ensures fidelity of initiation.
Mutations at TATA box reduce the accuracy of the startpoint of transcription of a gene.
2) GC-rich regions and CAAT boxes: located region between –40 and –110. Determine how frequently of the transcription event occurs by binding specific proteins. Mutations at these regions reduce the frequency of transcriptional starts 10 to 20 fold.
Gene Structure
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2-Regulatory regions : Enhancers and response elementsRegulate gene expression by binding with specific transcription factors.•Enhancers: bind the specific transcription factors (activators or transactivator) that increase the rate of transcription.
•Silencers or repressor: bind Other specific transcription factors (repressors) that depress the rate of transcription Enhancers and repressors: found in both upstream and downstream from the transcription site, which located hundreds or even thousands of bases from away the transcription unit. They also function in an orientation-independent fashion.
•
Gene Structure
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Phases of the transcription process: Pre-mRNA Synthesis
Initiation – promoter recognition and binding
Elongation–the actual process of ‘transcribing’ by RNA
polymerase II (5'→3' growing chain):
Termination–a sequence-dependent termination of RNA chain
growth:
Transcription
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Initiation: involved Formation of the basal transcription complex as
following: The general transcription factors (or basal factors at least
six) bind to the TATA box and facilitate the binding of RNA
polymerase II.1) The TATA-binding protein (TBP), a component of TFIID, binds to the
TATA box2) Transcription factors TFII A and B bind to TBP, then RNA polymerase
II binds to these factors and to DNA, and is aligned at the startpoint for transcription.
3) Then TFII E, F, and H bind, TFII H acts as ATP-dependent DNA helicase which is unwinding DNA for transcription. This intiation complex can transcribe at a basal level.
4) The rate of transcription can be increased by binding specific transcriptional (transactivators) to the enhancer and they interact with coactivator proteins of TFIID in the complex
Transcription
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Transcription Initiation – promoter recognition and binding
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Elongation–the actual process of ‘transcribing’ by RNA polymerase (5'→3' growing chain):
• RNA poly II recognize the startpoint and DNA
template, RNA poly II reads DNA 3'→5‘
and use the antisense strand (3'→5')o f DNA
as a template strand that is copied to produce
5'→3'RNA strand depending on the
Watson-Crick complementary.
• Do not need primer and catalyze
3'→5‘ phosphodiester bond
Transcription
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Elongation–the actual process of ‘transcribing’ by RNA polymerase (5'→3' growing chain):
• RNA polymerase II progresses along DNA
template leaving complex behind and
the initiation complex dissipates upon departure
of RNA polymerase.• RNA poly has constant synthesis rate about 30-40 nucleotides per second.
• RNA strand has exactly the same sequence as
the DNA 5'→3' sense strand, except that
the uracil base instead of thymine.
Transcription
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Termination–a sequence-dependent termination of RNA chain
growth:
• As RNA polymerase moves along the DNA template, reaching a 3'
termination sequence called the polyadenylation signal
(AAUAAA) . RNA polymerase stops and falls off the DNA
template strand. In the process, the pre-mRNA molecule is
released and the DNA strands re-form a double helix.
• Mutation at polyadenylation signal (AAUAAA) will reduce the
amount of mRNA.
Transcription
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RNA processing reactions: pre-mRNA (hnRNA)) convert
into mature mRNA:
1-Capping – addition of a 5´cap
•Began immediately after the initiation of RNA synthesis: by adding
a methylated guanosine (modified guanosine) to the 5’ end (leader
sequence) of the transcript by RNA poly II
•protects it from degradation by 5’-exonucleases during elongation
of RNA chain.
•increase the efficiency of translation of the mRNA by help the
transcript bind to the ribosome during protein synthesis
Transcription
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RNA processing reactions :
2-Tailing (polyadenylation) – addition of a 3´polyA tail
•Add poly A tail (up to 200 adenine nucleotides) to 3´ terminus by
poly A polymerase
•The poly (A) tail protects the mRNA from degradation by 3'
exonucleases.
•Help in mRNA export: the mature mRNA complexes with poly A-
binding protein and other proteins to migrate from nucleus into
cytoplasm through nuclear membrane pores.
Transcription
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RNA processing reactions :
3-Splicing – the removal of introns; the exons are ‘spliced together’:
Introns are removed and exons are spliced together to form the mature mRNA.
•Split site sequences: beginning (GU/5'splice donor) and ending (AG/ 3'splice
acceptor) of each intron, which are essential for splicing introns out of the
primary transcript.
•The exons joined together to form Open Reading Frame (ORF),which is coding
area specify amino acids
Transcription
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How can we interpret the presence of 100,000 kinds of mRNA that
resulting from25000 genes only?
One individual gene can produce different mRNAs coding to different proteins
due to:
•Different splicing products (Alternative splicing):
Alternative splicing represent in ability of genes to form multiple processed
mRNA contain different combinations of exons that coding to multiple proteins
•Use of different transcription initiation sites
Gene Expression
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Ribosomal RNA (rRNA): 18S, 28S, 5S, and 5.8S. • 18S, 28S, and 5.8S rRNA genes present in very many copies tandemly repeated
and expressed together, which transcribed into rRNA in Nucleolus by RNA polymerase I
• 5S rRNA produced by RNA polymerase III in the Nucleus. • rRNA comprise 80% of total RNA in the cell and associates with proteins to
form ribosomes Transfer RNA (tRNA): • tRNA genes are often multi-copy clusters expressed together, which
transcribed into tRNA by RNA polymerase III in the nucleus.• It has ability to carry the appropriate amino acid in the protein synthesis Messenger RNA (mRNA): • comprise about 5% of the total RNA and carries genetics information from
DNA for translation.• mRNA genes are single copy, which transcribed into mRNA in nucleus by
RNA polymerase II.
Types of RNA
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What are the differences between transcription in
mammalian and bacterial cells?
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THANK YOU